Method to amplify cardiac stem cells in vitro and in vivo

ABSTRACT

Compositions comprising stem cells delivered into infarcted myocardium by endocardial injection engraft and differentiate into myocytes, endothelial cells, and vascular smooth muscle, and do so without the requirement for survival enhancing modification. These cells engraft whether injected acutely (days) or late (months) after myocardial infarction, and the efficiency of engraftment correlates with the functional recovery of the heart. The stem cells also recruit endogenous cardiac precursor cells, reconstitute myocardial stem cell niches, and enhance endogenous cell differentiation into myocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a by-pass continuation-in-part, which claims thepriority of U.S. provisional patent application Nos. 60/976,663 and61/183,316 entitled “A METHOD TO AMPLIFY CARDIAC STEM CELLS IN VITRO ANDIN VIVO”, filed Oct. 1, 2007, and Jun. 2, 2009 respectively andPCT/US08/78379 filed Oct. 1, 2008 which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

Embodiments of the invention relate to stem cell compositions andmethods of tissue repair, and amplifying cardiac stem cells in vivo andin vitro.

BACKGROUND

Mesenchymal stem cells (MSCs) are bone marrow derived stem cells thathave entered clinical trials for the treatment of various diseasesincluding myocardial infarction and heart failure. In the past decadethere have been extensive attempts to characterize the nature of bonemarrow and cardiac precursor cell participation in recovery followingischemic injury to the heart. The idea that potentially reparativeprecursor cells exist in the marrow and in the heart has enormousclinical implications and has spurred a large number of clinical studiestesting whether bone marrow or its derivatives exert clinical recoveryfollowing myocardial infarction (MI) and other types of cardiac injury.Despite this work proceeding both at clinical and basic levels, noconsensus has emerged regarding the ability of these adult cell types todifferentiate into cellular elements comprising the heart. To thecontrary, there are diametrically opposing reports regarding this issue,and definitive proof of cellular engraftment is lacking in the clinicalsetting.

SUMMARY

This Summary is provided to present a summary of the invention tobriefly indicate the nature and substance of the invention. It issubmitted with the understanding that it will not be used to interpretor limit the scope or meaning of the claims.

This invention addresses a major limitation in the cardiac cell therapyarea. This limitation involves using cardiac stem cells to treatpatients with heart disease. Cardiac stem cells, while highly promisingas a therapy, require a tissue biopsy and a long period of growth in thelaboratory, MSCs can be used to accelerate the growth of CSCs in vitroor can be used to amplify CSCs in vivo, thereby eliminating the need forthe tissue biopsy.

In an preferred embodiment, a method of treating heart disease and heartdisorders in a patient comprises isolating stem cells from a patient ordonor; purifying the stem cells and obtaining mesenchymal stem cells;administering to a patient's cardiac tissue, mesenchymal stem cells in aconcentration effective to repair damaged cardiac tissue; and, treatingheart disease and heart disorders.

In another preferred embodiment, the mesenchymal stem cells areautologous or donor derived.

In another preferred embodiment, the mesenchymal stem cellsdifferentiate into multi-lineages. Preferably, the mesenchymal stemcells differentiate into at least one lineage of cardiac cells; morepreferably, the mesenchymal stem cells differentiate into at least twolineages of cardiac cells; more preferably, the mesenchymal stem cellsdifferentiate into three lineages of cardiac cells.

In another preferred embodiment, the lineages of cardiac cells areindentified by at least one marker comprising cardiac transcriptionfactor GATA-4; endothelial cell markers Factor VIII and KDR; vascularsmooth muscle marker α-smooth muscle actin; or cardiomyocyte markerα-sarcomeric actinin.

In another preferred embodiment, the stem cells are obtained from bonemarrow, circulation or tissues and organs.

In another preferred embodiment, the mesenchymal stem cells isolatedfrom adult bone marrow cells.

In another preferred embodiment, the mesenchymal cells recruitendogenous cardiac stem cells, reconstitute myocardial stem cell nichesand accelerate endogenous cell differentiation into myocytes.

In another preferred embodiment, the endogenous cardiac stem cells areindentified by at least one marker comprising connexin-43, N-cadherin,c-kit^(pos), CD3^(neg), CD14^(neg) and CD68^(neg).

In another preferred embodiment, cardiac stem cells are autologous ordonor derived.

In yet another preferred embodiment, the mesenchymal stem cell factorsare administered to the patient.

In one preferred embodiment, a method of recruiting endogenous cardiacstem cells to damaged heart tissue comprises administering to thedamaged heart tissue, purified mesenchymal stem cells; and, recruitingendogenous cardiac stem cells.

In a preferred embodiment, the mesenchymal stem cells are purified fromadult bone marrow.

In another preferred embodiment, the mesenchymal stem cells areautologous, heterologous, syngeneic, allogeneic or xenogeneic.

In another preferred embodiment, the damage to heart tissue can be fromany source or cause and comprises disease, physical damage, chemicaldamage, surgery, transplantation, or congenital defects.

In another preferred embodiment, the method of inducing and/oraccelerating cardiac stem cell proliferation comprises isolatingmesenchymal stem cells; co-culturing mesenchymal stem cells and cardiacstem cells in a concentration sufficient to induce and/or acceleratecardiac stem cells proliferation.

In another preferred embodiment, the cardiac stem cells differentiateinto cardiac cells expressing at least one of MDR1 or GATA-4.

In another preferred embodiment, the cardiac stem cells are derived froman autologous or histocompatible tissue biopsy.

In another preferred embodiment, the isolated the mesenchymal cells areadministered to a patient.

In another preferred embodiment, the mesenchymal cells and cardiac stemcells are autologous, heterologous, syngeneic, allogeneic or xenogeneic.

In yet another embodiment, the mesenchymal stem cells and cardiac stemcells are isolated from differing sources. Preferably, the cardiac stemcells are endogenous stem cells or donor derived. Cardiac stem cells arepreferably indentified by at least one marker comprising connexin-43,N-cadherin, c-kit^(pos), CD3^(neg), CD14^(neg) and CD68^(neg).

In another preferred embodiment, the mesenchymal cells are autologousstem cells and are administered to a patient in a therapeuticallyeffective dose to recruit endogenous stem cells to damaged tissue.

In another preferred embodiment, the mesenchymal stem cell andendogenous cardiac stem cells are optionally isolated and culturedex-vivo and administered to a patient.

In another preferred embodiment, a method of treating damaged cardiactissue comprises administering mesenchymal stem cells to cardiac tissue;stimulating cardiac stem cell in vivo proliferation; and, treatingdamaged cardiac tissue.

In another preferred embodiment, soluble factors from culturedmesenchymal stem cells are administered to cardiac tissue. In apreferred embodiment, a mesenchymal stem cell factor stimulates theproliferation of cardiac stem cells in vivo.

In another preferred embodiment, an antibody or aptamer is specific,i.e. specifically binds a mesenchymal stem cell factor.

In another preferred embodiment, a mesenchymal stem cell factorstimulates the proliferation of cardiac stem cells in vitro.

In another preferred embodiment a composition comprises mesenchymal stemcells and cardiac stem cells. In another preferred embodiment, thecomposition comprises mesenchymal stem cells from one source, e.g.autologous, donor derived, etc; and/or cardiac stem cells from anothersource, e.g. autologous, donor derived etc.

In another preferred embodiment, a mesenchymal stem cell comprises apolynucleotide encoding for a therapeutic agent, chemokine, growthfactor or ligands thereof.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1I are scans of photographs showing the trilineagedifferentiation of allogeneic MSCs. FIG. 1A: Cardiac lineage commitmentof GFP^(pos) αMSC expressing GATA-4 (arrows) in the BZ of a treatedheart 24 h after transplantation. FIG. 1B: Cardiac committed αMSCundergoing symmetric division in the infarct zone of a treated heart 72h after transplantation. FIG. 1C: GFP⁺ αMSC undergoing asymmetricdivision within the infarct zone of a 72 h treated heart and giving riseto a cardiac committed (GATA-4^(pos)) and an uncommitted daughter cell.FIG. 1D: Endothelial lineage commitment of αMSCs 72 h aftertransplantation, indicated by the colocalization of GFP and KDR/factorVIII (arrows). FIG. 1E: Magnification of the lower right corner of FIG.1C. FIG. 1F: vascular smooth muscle cell commitment of αMSCs 72 h aftertransplantation, as indicated by the co-localization of GFP withα-smooth muscle actin (arrows). FIG. 1G: Magnification of the lowerright corner of FIG. 1E. Electrical coupling of αMSCs with surroundingcardiac tissue 24 h after transplantation, as indicated by theco-localization of GFP and Connexin-43. FIG. 1I: GFP^(pos) cells in aregenerating zone (Rgz) 72 h after transplantation are expressingα-sarcomeric actinin (arrows) and are mechanically coupled by N-cadherinwith the infarcted (IZ) and border zone (BZ).

FIGS. 2A to 2J are scans of photographs showing the regeneration ofcardiac muscle, vasculature and stem cell niches. FIG. 2A: An immatureGFP^(pos) cardiac progenitor MSC expressing GATA-4 in the border zone ofa treated heart 2 weeks after transplantation. Notice the adjoiningc-kit^(pos) CSC. FIG. 2B: Immature MSCs in the border zone of a 2 weektreated heart, expressing α-sarcomeric actinin (arrows). FIG. 2C: Robustregeneration of the infarcted myocardium 2 weeks after transplantation,as demonstrated by the presence of Y-chromosome containing cardiacmyocytes. FIG. 2D: New coronary vessel formation 2 weeks aftertransplantation, as indicated by the Y-chromosome containing endothelialcell (arrow). FIG. 2E: The chimeric myocardium consisted of maturecardiac myocytes, cardiac precursors and immature MSCs, all connected byconnexin-43 with each other and the surrounding cardiac muscle (arrows).FIG. 2F: Definite cardiac myocyte differentiation of MSCs 2 weeks aftertransplantation, as indicated by the existence of Y-chromosomecontaining mature cardiac myocytes. FIG. 2G: Mobilization of c-kitposcells at the sites of injury 2 weeks after transplantation. C-kit cellswere found in clusters in close proximity to GFP cells. FIG. 2H: Incontrast to the αMSCs treated animals, Placebo and control groups,showed significantly lower numbers of c-kit cells which were alsoisolated. FIGS. 2I, 2J: The mobilized c-kit cells, often were connectedwith N-cadherin and connexin-43 with each other, the surrounding cardiactissue and with GFP^(pos) cells, indicating cardiac stem cell nichesreconstitution.

FIGS. 3A to 3J show the long-term functional recovery following αMSCstransplantation. FIG. 3A is a graph showing the assessment of global LVfunction by cine-MR images showing significant increase in % ejectionfraction in the αMSCs-treated group compared to placebo, at 2 months[16.9±1% increase in αMSCs, n=6, vs. 0.4±3.7% increase in placebo, n=4;*p=0.005] and 3 months after treatment [16.5±8% increase in αMSCs, n=6,vs. 5.5±4.6% decrease in placebo, n=4; ^(†)p=0.05]. FIGS. 3B, 3C arescans of photographs showing delayed contrast enhancement (DCE) MRimages 3 months after experimental myocardial infarction (MI) (FIG. 3B)and same short axis view after 3 months of αMSCs therapy (FIG. 3C).Notice the development of new endocardial tissue at the BZ of thetreated hearts (arrowheads). FIG. 3D is a graph showing the quantitationof infarct size by DCE MRI which showed a significant % decrease in scarsize in αMSCs animals compared to placebo group at 2 months [21.9±7.3%decrease in αMSCs, n=6, vs. 1.1±5.5% increase in placebo n=4; *p=0.047and 3 months after treatment [29.0±15.1% decrease in αMSCs, n=6, vs.4.3±9.4% increase in placebo, n=4; ^(†)p=0.01]. FIGS. 3E, 3F are scansof photographs showing representative 4 mm-thick slices from theharvested hearts 12 weeks after MI, illustrating differences in LVdiameter as well as in the extent of the scar size between the placeboand αMSCs-treated animals (FIGS. 3E and 3F respectively). FIG. 3G is agraph showing a plot of the peak circumferential strain (peak Ecc) ofthe endocardial wall over time, demonstrating significant improvementsin regional contractility of the treated-hearts (negative peak Eccdenotes normal regional contraction), 8 and 12 weeks after αMSCs therapy(*p=0.001 vs. placebo). FIG. 3H is a graph showing improvement incontractility, evidenced by the absolute changes in peak Ecc, exhibitedcorrelation with the absolute reduction in infarct size (Pearson'scorrelation: R=−0.97). FIG. 3I is a graph showing recovery in peak Eccwas strongly related to the αMSCs engraftment (R=−0.82). FIG. 3J is ascan of a photograph showing evidence of chimeric heart 12 weeks aftertransplantation, demonstrated by the presence of newly formedGATA-4^(pos)/Y^(pos) mature CM.

FIGS. 4A to 4E show the regeneration of large and small coronary vesselsfollowing αMSC transplantation. FIGS. 4A-4C are scans of photographsshowing confocal microscopy of large (FIG. 4A), middle-sized (FIG. 4B)and capillary vessels (FIG. 4C) in BZ of the αMSCs-treated groupexhibiting Y^(pos) cells into their structure. Large and middle-sizedregenerated vessels co-localized with a smooth muscle actin and factorVIII. FIGS. 4D, 4E are graphs showing quantification of new vesseldensities per unit volume of myocardium. The majority of the regeneratedvessels were detected in the BZ of the αMSCs treated hearts.

FIG. 5 is a schematic illustration showing s summary of the in vivoresults (panel insets a-f).

FIGS. 6A-6E show Laser Scanning Cytometry (LSC) for mapping celltrafficking. FIGS. 6A-6C: Within 72 h post implantation, the GFP^(pos)MSCs migrated from the endomyocardial site of injection to the infarctedsubepicardial rim. No cells could be detected into the healthysurrounding myocardium indicating that MSCs trafficking was driven fromdamage signals. FIGS. 6D, 6E: Confocal Microscopy for anti-GFP andY-chromosome was used to verify the detection of MSCs. Note that not allGFP^(pos) cells are Y-chromosome^(pos) and vice versa. The sensitivityof combined FISH/immunofluorescence detection was 45.5+2.1% of the totalmale cells.

FIGS. 7A to 7D are scans of photographs showing robust retention ofαMSCs two weeks after intramyocardial transplantation. FIG. 7A showshematoxylin and eosin histological stain demonstrating the tracing of aninjection site. FIG. 7B shows the presence of GFP^(pos) cells within thesame injection confirming the origin of the exogenous cells. FIGS. 7C,7D: Connexin-43 and β1-Integrin were used as markers of homing andengraftment of the MSCs in the host myocardium. Notice the strongpresence of c-kit cells in FIG. 7C.

FIGS. 8A to 8C show the fate of the allografts in the host myocardiumduring the first 72 h after transplantation. FIG. 8A is a scan of aphotograph showing immunofluorescence detection of the serine-10phosphorylated Histone H3 (arrows) demonstrated the presence of mitoticαMSCs within an injection site, 72 h after transplantation. FIG. 8B:Co-localization of phospho-H3 and cleaved caspase-3 illustrated thepremature chromatin condensation of an αMSC (arrow) and initiation ofapoptosis 72 h after transplantation. Arrowhead shows an adjacent αMSCin mitosis. FIG. 8C: The number of apoptotic MSCs was counterbalanced byan analogous number of cells undergoing mitosis. Mitotic:phopshoHistone-H3^(pos)-GFP^(pos): 1.2±0.7% of the total GFP^(pos) cellsat 24 h (n=1) and 1.3±0.4% at 72 h (n=3)]; Differentiating:GATA-4^(pos)-GFP^(pos) 13.4±5.4% of the GFPpos detected cells at 24 h(n=1) and 23.4±3.2%, at 72 hours (n=3); Apoptotic:caspase3^(pos)-GFP^(pos): 0.31±1.0% at 24 h (n=1) and 1.0±0.6% at 72 hof the total GFPpos cells (n=3).

FIGS. 9A to 9D show the mobilization of endogenous c-kit^(pos) CSCs 2weeks after transplantation of MSCs. FIGS. 9A-9C show the dramaticrecruitment of c-kit^(pos) cells at the sites of injection. The c-kitcells were in clusters in the IZ (FIG. 9A) and BZ (FIG. 9B) of thetreated hearts, but were mainly isolated in the healthy zones and/or thehearts of the nontreated animals (FIG. 9C). FIG. 9D is a graph showingthe distribution of the c-kit cells within the different zones of thetreated and untreated animals. IZ: 2.14±0.09 cells/mm² for the αMSCsgroup (n=3) vs. 0.04±0.02 cells/mm² for the placebo (n=3) and 0.09±0.04cells/mm² for the control groups (n=3) respectively, *p<0.001; BZ:0.74±0.12 cells/mm² for the αMSCs vs. 0.008±0.002 cells/mm² for theplacebo and 0.013±0.003 cells/mm² for the control groups respectively,^(†)p=0.001; RZ: 0.10±0.01 cells/mm² for the αMSCs vs. 0.002±0.001cells/mm² for the placebo and 0.01±0.005 cells/mm² for the controlgroups respectively, *p<0.001.

FIGS. 10A to 10C show the stimulation of endogenous cardiac repairfollowing αMSCs transplantation. FIGS. 10A, 10B: Besides the GFPposMSCs, the regenerated CSC niches were containing clusters of c-kit^(pos)cells which were co-localized with MDR1^(pos) and GATA-4^(pos). FIG.10C: Quantification of the c-kit^(pos)/GATA-4^(pos) cardiac precursorcells between groups, demonstrated an active endogenous repair processin the MSCs treated animals which was commenced, but not restricted, inthe BZ of the infarcted hearts. IZ: 0.4±0.08% of total c-kit^(pos) cellsin the αMSCs group (n=3) vs. 0.3±0.3% in the placebo (n=3) and none inthe control (n=3), p=0.5; BZ: 3.9±1.6% in the αMSCs group vs. 0.28±0.14%in the placebo and none in the control, *p=0.038; RZ: none for anygroups).

FIGS. 11A to 11C show the long-term retention of αMSCs. FIG. 11A:Transplanted αMSCs were found in clusters into the IZ and BZ of the hostmyocardium. Co-localization of BrdU with Y chromosome were used toconfirm the origin of the allografts (arrows). Some of these cells hadlost BrdU, presumably by mitotic division (arrowheads). FIG. 11B:Distribution of the αMSCs in the chronic infarcted myocardium. Cellswere detected in the IZ and BZ but not the remote healthy zones. FIG.11C: Newly formed cardiomyocytes were fully functional and integratedinto the host myocardium; Demonstration of electrical coupling of αMSCswith the resident cardiomyocytes via the development of connexin-43 gapjunctions (arrows).

FIGS. 12A-12D: Assessment of infarct size and global LV function bycardiac MRL (FIG. 12A) Significant decrease in the absolute value of theinfarct size between MSCs and CCM-treated groups could be documented asearly as 4 days post injections. By 8 weeks MSCs group exhibiteddiminished scar size (p<0.001) [absolute decrease (% of LV) 8.3±1.8% vs.0.7±0.9% at day 4 (*p=0.018); 10.9±1.4% vs 3.3±1.7% at 2 weeks(*p=0.002); and 13±0.6% vs 2.3±1.3% at 8 weeks post injection (*p=0.002)between MSCs and CCM groups respectively]. (FIG. 12B) Ejection fractionwas similar between the two groups through the 2 month study. However,by 2 weeks the MSCs group exhibited a significant recovery compared topost-MI (absolute decrease in EF between baseline and pre injection:8.9±1.7% vs 12.3±3.7% (p=NS); baseline and 4d post-injection: 3.5±4.6%vs 11.1±2.4% (p=NS); baseline and 2 weeks post injection: 0.72±5.6 vs9.2±2.5% (p=NS); baseline and 8 weeks post injection: 1.03±11.5% vs.10.5±3.4% (p=NS) between MSCs and CCM-group respectively]. (†p=0.042 and†p=0.026 within MSCs group at 2 and 8 weeks respectively). Blue arrowsindicate the day before injections. (FIGS. 12C, 12D) Delayed contrasthyperenhanced images of MSCs (FIG. 12C) and CCM (FIG. 12D) treatedanimals before and 8 weeks after injections. Notice the reduction ininfarct size (yellow) in the MSCs but not the CCM-treated heart. Meanvalues±SEM (n=6 each at baseline, 4-days and 2 weeks, n=3 each at 8weeks).

FIGS. 13A-13E: MSCs differentiate into new cardiac myocytes and vessels.(FIG. 13A) Masson's trichrome stained tissue section showing the contextof a Y-chromosome containing region with respect to the infarct, 2 weeksafter MSCs therapy. MSCs differentiated into new myocardial tissue atthe border line of a previously infarcted region. The section is located˜8 mm far from the apex and ˜30 mm from base of the LV. (FIG. 13B)Chimeric myocardium as indicated by the Y-chromosome containing myocytesin the cross-section of panel (FIG. 13A). (FIG. 13C) New coronary vesselformation 2 weeks after transplantation, as indicated by theY-chromosome containing endothelial cell (arrow). (FIG. 13D) Chimericmyocardium consisted of mature cardiac myocytes (arrow), cardiacprecursors (arrowhead) and immature MSCs (open arrow), all connected byconnexin-43 with each other and the surrounding cardiac muscle. (FIG.13E) Y-chromosome containing mature cardiomyocytes highlighted byLaminin (open arrows). [MI, myocardial infarct; LV, left ventricle; RV,right ventricle].

FIGS. 14A-14H. MSCs stimulate endogenous CPCs. (FIG. 14A)Differentiation of MSCs peaks 3-days postimplantation and theregenerated allografts are sustained for 2 months after transplantation[(see FIG. 16 for 24 h and 72 h values of GFP⁺/GATA-4⁺ MSCs);Y-chromosome⁺ cardiomyocytes/cm³: 75.3±24.9 in IZ and 135.5±64.1 in BZat 2 weeks, and 76.7±33.2 in IZ and 185.02±64.3 in BZ at 8 weeks]. Twoweeks later, MSCs stimulated a dramatic expansion of the endogenousc-kit⁺ CPCs pool. (FIG. 14B) Quantification of c-kit⁺/GATA-4⁺ CPCsillustrated a significantly increased commitment of CPCs in theMSC-group. IZ: 6.8±1.7 GATA-4⁺ CPCs/cm³ in the MSCs (n=6) vs. 2.9±2.9CPCs/cm³ in the placebo (n=3), none in the control (n=3), and 0.5±0.5CPCs/cm³ in the CCM groups respectively, ‡p=0.019; BZ: 15.1±3.1 GATA-4⁺CPCs/cm³ in the MSCs vs. 1.0±1.0 CPCs/cm³ in the placebo, none in thecontrol (n=3), and 0.7±0.7 CPCs/cm³ in the CCM groups respectively,†p<0.0001; RZ: none for any groups). (FIGS. 14C, 14D) Co-localization ofGFP with GATA-4 (FIG. 14C) and a-sarcomeric actinin (FIG. 14D) documentscardiac precursors of MSCs origin in the infarcted hearts which arefound in close proximity to c-kit⁺ CPCs (FIG. 14C). (FIG. 14E) Clusterof c-kit+ CPCs in an MSCs-treated heart; numerous CPCs are committed tocardiac lineage documented by GATA-4 and MDR-1 co-expression (arrows).(FIG. 14F) c-kit⁺ cells were found isolated in non-MSC treated animals.(FIGS. 14G, 14H) MSCs interact with c-kit⁺ CPCs by connexin-43 (FIG.14F) and N-cadherin (FIG. 14G) connections, closely resembling cardiacstem cell niches.

FIGS. 15A-15K ₁. Ex-vivo cardiac stem cell niche regeneration. (FIG.15A) Culture of biopsies in a lawn of MSCs for 1 week facilitates adramatic outgrowth of c-kit⁺ CPCs. A mean of 19,158.82±6,505.7 vs3,347.05±1,519.5 c-kit⁺ cells were purified from biopsies cultured withand without MSCs respectively (*p=0.003). (FIG. 15B) A small number ofcells outgrow from the biopsies cultured alone. (FIG. 15C) Organotypiccultures with MSCs have become confluent while, some GFP⁺ MSCs haveinfiltrated the heart samples. (FIG. 15D, FIG. 15E). In contrast to thepurified c-kit+ cells from the biopsy-alone which are large, quiescentcells with a macrophages morphology (FIG. 15E), co-cultures with MSCsegress small, semi-adherent CPCs that renew their population constantly(FIG. 15D). (FIGS. 15F, 15G), Immunostaining of the primary cellcultures documents interactions between GFP⁺ MSCs and c-kit⁺ cells asindicated by co-localization with CCM-43; these clusters closelyresemble cardiac stem cell niches. (FIGS. 15H-15H ₃) Cytospins ofpurified c-kit⁺ CPCs, illustrating co-localization with MDR1, while lackof the surface marker CD68 from the vast majority of them excludes aninflammatory or mast cell phenotype. (FIG. 15I) Spontaneouslycontracting CPCs 3 days after co-culture in a transwell insert withNRCMs. Arrows indicate 8 contractions of the CPCs through a 12 secperiod of time. (FIGS. 15J-15J ₂) Expression of is 1-1 in a subset ofthe CPCs. This information underlies that mechanisms of cellreprogramming (perhaps reactivation of the fetal gene program) areimplicated in the MSCs-mediated recruitment of c-kit⁺ CPCs. (FIGS.15K-15K ₁) Nkx2-5, is expressed in more than 90% of the CPCs. Meanvalues *SEM (n=19 each).

FIGS. 16A-16D. Fate of the allografts during the first 3 days aftertransplantation. (FIG. 16) The numbers of GFP⁺ cells that were detectedin the porcine LVs did not differ significantly between the first 24 and72 h post-implantation. Histological quantification revealed that the100×10⁶ transplanted MSCs accounted for 1585.17±746.7 cells/cm³ at 24 hand 1317.1±393.3 cells/cm³ at 72 h. The extent of differentiating,mitotic and apoptotic MSCs during the first 3 days: myocytic(GFP⁺/GATA-4⁺): 63.7±23.9 cells/cm³ at 24 h and 198.03±36.3 cells/cm³ at72 h; angiogenic (GFP⁺/Factor VIII ⁺): none at 24 h, 0.94±0.4 cells/cm³at 72 h; mitotic: phospho-H3⁺/GFP⁺: 0.8±0.5 cells/cm³ at 24 h and1.2±0.4 cells/cm³ at 72 h]; apoptotic: activated caspase3⁺/GFP⁺: 0.2±0.2cells/cm³ at 24 h and 1.0±0.6 cells/cm³ at 72 h. (FIG. 16B) The presenceof exogenous cells within the host myocardium was confirmed by both GFPand chromosome co-localization. (FIG. 16C) Co-localization of phospho-H3with GFP demonstrates the presence of mitotic MSCs within an injectionsite, 72 h after transplantation. (FIG. 16D) Colocalization ofphospho-H3 and cleaved caspase-3 illustrates premature chromatincondensation of an MSC and initiation of apoptosis 72 h aftertransplantation. Arrow shows an adjacent MSC in mitosis. Mean Values±SEM(n=2 at 24 h, n=3 at 72 h).

FIGS. 17A-17F: MSCs stimulate amplification of endogenous c-kit+ CPCs 2weeks after injection. (FIG. 17A) Recruitment of c-kit⁺ CPCs in theMSCs-treated vs non-treated hearts and distribution of the c-kit cellswithin the different zones. IZ: 897.9+195.2 c-kit⁺ cells/cm³ for theMSCs group (n=6) vs. 78.1±32.6 cells/cm³ for the placebo (n=3),69.4±21.2 cells/cm³ for the control and 27.1±18 cells/cm³ for the Cxgroups (n=3) respectively, *p<0.001; BZ: 467.5±70.6 cells/cm³ for theMSCs vs. 18.9±5.9 cells/cm³ for the placebo, 24.1±8.4 cells/cm³ for thecontrol and 16.1±11.3 cells/cm³ for the Cx groups respectively,*p=0.001; RZ: 87.5±17.9 c-kit⁺ cells/cm³ for the MSCs vs. 0.5±0.5cells/cm³ for the placebo, 7.4±3.8 cells/cm³ for the control and nonefor the Cx groups respectively, *p<0.001. (FIG. 17B) Endogenous c-kit⁺CPCs become functionally coupled with the infarcted myocardium asindicated by colocalization with N-cadherin (arrows). (FIG. 17C), Alarge cluster of c-kit⁺ CPCs connected to each other and to adjacentGFP⁺ MSCs by connexin-43. (FIGS. 17D-17F), Representative figuresillustrating the non-inflammatory/mast cell phenotype of the c-kit+CPCs. While clusters of CD68^(pos)/ckit^(neg) cells could be detected inthe non MSCs-treated hearts (FIG. 17C), the MSCs-treated hearts wererich in c-kit^(pos)/CD-68^(neg) and CD3^(neg) clusters of CPCs (FIG.17D) Mean values±SEM (n=6 MSCs, 3 placebo, 3 Cx and 3 Control).

DETAILED DESCRIPTION

The present invention is described with reference to the attachedfigures, wherein like reference numerals are used throughout the figuresto designate similar or equivalent elements. The figures are not drawnto scale and they are provided merely to illustrate the instantinvention. Several aspects of the invention are described below withreference to example applications for illustration. It should beunderstood that numerous specific details, relationships, and methodsare set forth to provide a full understanding of the invention. Onehaving ordinary skill in the relevant art, however, will readilyrecognize that the invention can be practiced without one or more of thespecific details or with other methods. The present invention is notlimited by the illustrated ordering of acts or events, as some acts mayoccur in different orders and/or concurrently with other acts or events.Furthermore, not all illustrated acts or events are required toimplement a methodology in accordance with the present invention.

Embodiments of the invention may be practiced without the theoreticalaspects presented. Moreover, the theoretical aspects are presented withthe understanding that Applicants do not seek to be bound by the theorypresented.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As used herein, “heart disease” refers to any type of heart diseaseincluding cardiomyopathy, hypertrophic cardiomyopathy, dilatedcardiomyopathy, atherosclerosis, coronary artery disease, ischemic heartdisease, myocarditis, viral infection, wounds, hypertensive heartdisease, valvular disease, congenital heart disease, myocardialinfarction, congestive heart failure, arrhythmias, diseases resulting inremodeling of the heart, etc. Diseases of the heart can be due to anyreason, such as for example, damage to cardiac tissue such as a loss ofcontractility (e.g., as might be demonstrated by a decreased ejectionfraction).

Cardiac damage or disorder characterized by insufficient cardiacfunction includes any impairment or absence of a normal cardiac functionor presence of an abnormal cardiac function. Abnormal cardiac functioncan be the result of disease, injury, and/or aging. A used herein,abnormal cardiac function includes morphological and/or functionalabnormality of a cardiomyocyte, a population of cardiomyocytes, or theheart itself. Non-limiting examples of morphological and functionalabnormalities include physical deterioration and/or death ofcardiomyocytes, abnormal growth patterns of cardiomyocytes,abnormalities in the physical connection between cardiomyocytes, under-or over-production of a substance or substances by cardiomyocytes,failure of cardiomyocytes to produce a substance or substances whichthey normally produce, and transmission of electrical impulses inabnormal patterns or at abnormal times. Abnormalities at a more grosslevel include dyskinesis, reduced ejection fraction, changes as observedby echocardiography (e.g., dilatation), changes in EKG, changes inexercise tolerance, reduced capillary perfusion, and changes as observedby angiography. Abnormal cardiac function is seen with many disordersincluding, for example, ischemic heart disease, e.g., angina pectoris,myocardial infarction, chronic ischemic heart disease, hypertensiveheart disease, pulmonary heart disease (cor pulmonale), valvular heartdisease, e.g., rheumatic fever, mitral valve prolapse, calcification ofmitral annulus, carcinoid heart disease, infective endocarditis,congenital heart disease, myocardial disease, e.g., myocarditis, dilatedcardiomyopathy, hypertensive cardiomyopathy, cardiac disorders whichresult in congestive heart failure, and tumors of the heart, e.g.,primary sarcomas and secondary tumors. Heart damage also includeswounds, such as for example, knife wound; biological (e.g. viral;autoimmune diseases) or chemical (e.g. chemotherapy, drugs); surgery;transplantation and the like.

“Myocardial ischemia” refers to a lack of oxygen flow to the heart whichresults in myocardial ischemic damage. As used herein, the phrasemyocardial ischemic damage includes damage caused by reduced blood flowto the myocardium. Non-limiting examples of causes of myocardialischemia and myocardial ischemic damage include: decreased aorticdiastolic pressure, increased intraventricular pressure and myocardialcontraction, coronary artery stenosis (e.g., coronary ligation, fixedcoronary stenosis, acute plaque change (e.g., rupture, hemorrhage),coronary artery thrombosis, vasoconstriction), aortic valve stenosis andregurgitation, and increased right atrial pressure. Non-limitingexamples of adverse effects of myocardial ischemia and myocardialischemic damage include: myocyte damage (e.g., myocyte cell loss,myocyte hypertrophy, myocyte cellular hyperplasia), angina (e.g., stableangina, variant angina, unstable angina, sudden cardiac death),myocardial infarction, and congestive heart failure. Damage due tomyocardial ischemia may be acute or chronic, and consequences mayinclude scar formation, cardiac remodeling, cardiac hypertrophy, wallthinning, dilatation, and associated functional changes. The existenceand etiology of acute or chronic myocardial damage and/or myocardialischemia may be diagnosed using any of a variety of methods andtechniques well known in the art including, e.g., non-invasive imaging(e.g., MRI, echocardiography), angiography, stress testing, assays forcardiac-specific proteins such as cardiac troponin, and clinicalsymptoms. These methods and techniques as well as other appropriatetechniques may be used to determine which subjects are suitablecandidates for the treatment methods described herein.

“Stem cell niche” refers to the microenvironment in which stem cells arefound, which interacts with stem cells to regulate stem cell fate. (See,for example, Kendall Powell, Nature 435, 268-270 (2005). The word‘niche’ can be in reference to the in vivo or in vitro stem cellmicroenvironment. During embryonic development, various niche factorsact on embryonic stem cells to alter gene expression, and induce theirproliferation or differentiation for the development of the fetus.Within the human body, stem cell niches maintain adult stem cells in aquiescent state, but after tissue injury, the surroundingmicroenvironment actively signals to stem cells to either promote selfrenewal or differentiation to form new tissues. Several factors areimportant to regulate stem cell characteristics within the niche:cell-cell interactions between stem cells, as well as interactionsbetween stem cells and neighboring differentiated cells, interactionsbetween stem cells and adhesion molecules, extracellular matrixcomponents, the oxygen tension, growth factors, cytokines, andphysiochemical nature of the environment including the pH, ionicstrength (e.g. Ca²⁺ concentration, metabolites like ATP are alsoimportant. The stem cells and niche may induce each other duringdevelopment and reciprocally signal to maintain each other duringadulthood. The niche also refers to specific anatomic locations thatregulate how they participate in tissue generation, maintenance andrepair. The niche saves stem cells from depletion, while protecting thehost from over-exuberant stem-cell proliferation. It constitutes a basicunit of tissue physiology, integrating signals that mediate the balancedresponse of stem cells to the needs of organisms. Yet the niche may alsoinduce pathologies by imposing aberrant function on stem cells or othertargets. The interplay between stem cells and their niche creates thedynamic system necessary for sustaining tissues, and for the ultimatedesign of stem-cell therapies.

“Biological samples” include solid and body fluid samples. Thebiological samples used in the present invention can include cells,protein or membrane extracts of cells, blood or biological fluids suchas ascites fluid or brain fluid (e.g., cerebrospinal fluid). Examples ofsolid biological samples include, but are not limited to, samples takenfrom tissues of the central nervous system, bone, breast, kidney,cervix, endometrium, head/neck, gallbladder, parotid gland, prostate,pituitary gland, muscle, esophagus, stomach, small intestine, colon,liver, spleen, pancreas, thyroid, heart, lung, bladder, adipose, lymphnode, uterus, ovary, adrenal gland, testes, tonsils and thymus. Examplesof “body fluid samples” include, but are not limited to blood, serum,semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bonemarrow, lymph, and tears.

“Bone marrow derived progenitor cell” (BMDC) or “bone marrow derivedstem cell” refers to a primitive stem cell with the machinery forself-renewal constitutively active. Included in this definition are stemcells that are totipotent, pluripotent and precursors. A “precursorcell” can be any cell in a cell differentiation pathway that is capableof differentiating into a more mature cell. As such, the term “precursorcell population” refers to a group of cells capable of developing into amore mature cell. A precursor cell population can comprise cells thatare totipotent, cells that are pluripotent and cells that are stem celllineage restricted (i.e. cells capable of developing into less than allhematopoietic lineages, or into, for example, only cells of erythroidlineage). As used herein, the term “totipotent cell” refers to a cellcapable of developing into all lineages of cells. Similarly, the term“totipotent population of cells” refers to a composition of cellscapable of developing into all lineages of cells. Also as used herein,the term “pluripotent cell” refers to a cell capable of developing intoa variety (albeit not all) lineages and are at least able to developinto all hematopoietic lineages (e.g., lymphoid, erythroid, andthrombocytic lineages). Bone marrow derived stem cells contain twowell-characterized types of stem cells. Mesenchymal stem cells (MSC)normally form chondrocytes and osteoblasts. Hematopoietic stem cells(HSC) are of mesodermal origin that normally give rise to cells of theblood and immune system (e.g., erythroid, granulocyte/macrophage,magakaryocite and lymphoid lineages). In addition, hematopoietic stemcells also have been shown to have the potential to differentiate intothe cells of the liver (including hepatocytes, bile duct cells), lung,kidney (e.g., renal tubular epithelial cells and renal parenchyma),gastrointestinal tract, skeletal muscle fibers, astrocytes of the CNS,Purkinje neurons, cardiac muscle (e.g., cardiomyocytes), endothelium andskin.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to whom it is later to be re-introducedinto the individual.

The term “xenogeneic cell” refers to a cell that derives from adifferent animal species than the animal species that becomes therecipient animal host in a transplantation or vaccination procedure.

The term “allogeneic cell” refers to a cell that is of the same animalspecies but genetically different in one or more genetic loci as theanimal that becomes the “recipient host”. This usually applies to cellstransplanted from one animal to another non-identical animal of the samespecies.

The term “syngeneic cell” refers to a cell which is of the same animalspecies and has the same genetic composition for most genotypic andphenotypic markers as the animal who becomes the recipient host of thatcell line in a transplantation or vaccination procedure. This usuallyapplies to cells transplanted from identical twins or may be applied tocells transplanted between highly inbred animals.

The terms “patient” or “individual” are used interchangeably herein, andrefers to a mammalian subject to be treated, with human patients beingpreferred. In some cases, the methods of the invention find use inexperimental animals, in veterinary application, and in the developmentof animal models for disease, including, but not limited to, rodentsincluding mice, rats, and hamsters; and primates.

“Diagnostic” or “diagnosed” means identifying the presence or nature ofa pathologic condition. Diagnostic methods differ in their sensitivityand specificity. The “sensitivity” of a diagnostic assay is thepercentage of diseased individuals who test positive (percent of “truepositives”). Diseased individuals not detected by the assay are “falsenegatives.” Subjects who are not diseased and who test negative in theassay, are termed “true negatives.” The “specificity” of a diagnosticassay is 1 minus the false positive rate, where the “false positive”rate is defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology or symptoms of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. As used herein, “ameliorated” or“treatment” refers to a symptom which is approaches a normalized value(for example a value obtained in a healthy patient or individual), e.g.,is less than 50% different from a normalized value, preferably is lessthan about 25% different from a normalized value, more preferably, isless than 10% different from a normalized value, and still morepreferably, is not significantly different from a normalized value asdetermined using routine statistical tests.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptide oraptamers, means that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody. Thus, an antibody that “specificallybinds to” or is “specific for” a particular polypeptide or an epitope ona particular polypeptide is one that binds to that particularpolypeptide or epitope on a particular polypeptide without substantiallybinding to any other polypeptide or polypeptide epitope.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, embryology, andcardiophysiology.

With respect to tissue culture and embryonic stem cells, the reader maywish to refer to Teratocarcinomas and embryonic stem cells: A practicalapproach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide toTechniques in Mouse Development (P. M. Wasserman et al. eds., AcademicPress 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles,Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic StemCells: Prospects for Application to Human Biology and Gene Therapy (P.D. Rathjen et al., Reprod Fertil. Dev. 10:31, 1998). With respect to theculture of heart cells, standard references include The Heart Cell inCulture (A. Pinson ed., CRC Press 1987), Isolated Adult Cardiomyocytes(Vols. I & II, Piper & Isenberg eds., CRC Press 1989), Heart Development(Harvey & Rosenthal, Academic Press 1998), I Left my Heart in SanFrancisco (T. Bennet, Sony Records 1990); and Gone with the Wnt (M.Mitchell, Scribner 1996).

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, hivitrogen,Sigma-Aldrich, and ClonTech.

Stem Cell Compositions

Embodiments of the invention provide for compositions comprisingmesenchymal stem cells (MSCs) utilized, in some aspects, for theacceleration of the preparation of cardiac stem cells (CSCs.) Enhancingthe CSCs in vitro will improve their utility. MSCs can also be used toproliferate CSCs in vivo. The CSCs proliferate and differentiate intocardiac cells, e.g. cardiomyocytes. Briefly, the results describedherein show that injections of MSCs into pig hearts caused massiveproliferation of CSCs. MSCs bind to and form complexes with CSCs, andcause their proliferation and differentiation into cardiac myocytes.Without wishing to be bound by theory, the cardiac stem cellproliferation can be due to a mesenchymal stem cell factor, a receptorligand interaction between the MSCs and CSCs or other cells in thecardiac tissue, or combinations of both.

In order to determine whether MSCs had cardiac precursor potential orwhether their therapeutic effect is exerted through paracrine effects,GFP labeled male allogeneic adult bone marrow derived mesenchymal stemcells (αMSCs) were injected into female pigs following myocardialinfarction (MI) (FIG. 5). It was hypothesized that allogeneic adult bonemarrow derived mesenchymal stem cells (αMSCs) were true adult precursorcells which could differentiate into three cardiac cell lineages, andthat αMSCs participate in cardiac recovery by forming cell-cellinteractions with existing myocardial elements including with endogenousprecursor cells.

A stem cell is a cell from the embryo, fetus, or adult that has, undercertain conditions, the ability to reproduce itself for long periods or,in the case of adult stem cells, throughout the life of the organism. Italso can give rise to specialized cells that make up the tissues andorgans of the body.

A pluripotent stem cell has the ability to give rise to types of cellsthat develop from the three germ layers (mesoderm, endoderm, andectoderm) from which all the cells of the body arise. The only knownsources of human pluripotent stem cells are those isolated and culturedfrom early human embryos and from fetal tissue that was destined to bepart of the gonads.

An embryonic stem cell is derived from a group of cells called the innercell mass, which is part of the early (4- to 5-day) embryo called theblastocyst. Once removed from the blastocyst the cells of the inner cellmass can be cultured into embryonic stem cells. These embryonic stemcells are not themselves embryos.

An adult stem cell is an undifferentiated (unspecialized) cell thatoccurs in a differentiated (specialized) tissue, renews itself, andbecomes specialized to yield all of the specialized cell types of thetissue in which it is placed when transferred to the appropriate tissue.Adult stem cells are capable of making identical copies of themselvesfor the lifetime of the organism. This property is referred to as“self-renewal.” Adult stem cells usually divide to generate progenitoror precursor cells, which then differentiate or develop into “mature”cell types that have characteristic shapes and specialized functions,e.g., muscle cell contraction or nerve cell signaling. Sources of adultstem cells include bone marrow, blood, the cornea and the retina of theeye, brain, skeletal muscle, dental pulp, liver, skin, the lining of thegastrointestinal tract and pancreas.

Stem cells from the bone marrow are the most-studied type of adult stemcells. They can be used clinically to restore various blood and immunecomponents to the bone marrow via transplantation. There are currentlyidentified two major types of stem cells found in bone marrow:hematopoietic stem cells (HSC, or CD34⁺ cells) which are typicallyconsidered to form blood and immune cells, and stromal (mesenchymal)stem cells (MSC) that are typically considered to form bone, cartilage,muscle and fat. However, both types of marrow-derived stem cellsrecently have demonstrated extensive plasticity and multipotency intheir ability to form the same tissues.

The marrow, located in the medullary cavity of bones, is the sole siteof hematopoiesis in adult humans. It produces about six billion cellsper kilogram of body weight per day. Hematopoietically active (red)marrow regresses after birth until late adolescence after which time itis focused in the lower skull vertebrae, shoulder and pelvic girdles,ribs, and sternum. Fat cells replace hematopoietic cells in the bones ofthe bands, feet, legs and arms (yellow marrow). Fat comes to occupyabout fifty percent of the space of red marrow in the adult and furtherfatty metamorphosis continues slowly with aging. In very oldindividuals, a gelatinous transformation of fat to a mucoid material mayoccur (white marrow). Yellow marrow can revert to hematopoieticallyactive marrow if prolonged demand is present such as with hemolyticanemia. Thus hematopoiesis can be expanded by increasing the volume ofred marrow and decreasing the development (transit) time from progenitorto mature cell.

The marrow stroma consists principally of a network of sinuses thatoriginate at the endosteum from cortical capillaries and terminate incollecting vessels that enter the systemic venous circulation. Thetrilaminar sinus wall is composed of endothelial cells; anunderdeveloped, thin basement membrane, and adventitial reticular cellsthat are fibroblasts capable of transforming into adipocytes. Theendothelium and reticular cells are sources of hematopoietic cytokines.Hematopoiesis takes place in the intersinus spaces and is controlled bya complex array of stimulatory and inhibitory cytokines, cell-to-cellcontacts and the effects of extracellular matrix components on proximatecells. In this unique environment, lymphohematopoietic stem cellsdifferentiate into all of the blood cell types. Mature cells areproduced and released to maintain steady state blood cell levels. Thesystem may meet increased demands for additional cells as a result ofblood loss, hemolysis, inflammation, immune cytopenias, and othercauses.

A “progenitor or precursor” cell occurs in fetal or adult tissues and ispartially specialized; it divides and gives rise to differentiatedcells. Researchers often distinguish precursor/progenitor cells fromadult stem cells in that when a stem cell divides; one of the two newcells is often a stem cell capable of replicating itself again. Incontrast when a progenitor/precursor cell divides, it can form moreprogenitor/precursor cells or it can form two specialized cells.Progenitor/precursor cells can replace cells that are damaged or dead,thus maintaining the integrity and functions of a tissue such as liveror brain.

Mesenchymal stem cells are the formative pluripotential blast cellsfound inter alia in bone marrow, blood, dermis and periosteum that arecapable of differentiating into any of the specific types of mesenchymalor connective tissues (i.e. the tissues of the body that support thespecialized elements; particularly adipose, osseous, cartilaginous,elastic, and fibrous connective tissues) depending upon variousinfluences from bioactive factors, such as cytokines.

The isolation and purification of mesenchymal stem cells has beendescribed in detail in the examples which follow. In one preferredembodiment, mesenchymal stem cells are isolated from bone marrow ofadult patients. In one aspect, the cells are passed through a densitygradient to eliminate undesired cell types. The cells are preferably,plated and cultured in appropriate media. In another preferredembodiment, the cells are cultured for at least one day, preferably,about three to about seven days, and removing non-adherent cells. Theadherent cells are plated and expanded.

Other means for isolating and culturing stem cells useful in the presentinvention are well known. Umbilical cord blood is an abundant source ofhematopoietic stem cells. The stem cells obtained from umbilical cordblood and those obtained from bone marrow or peripheral blood appear tobe very similar for transplantation use. Placenta is an excellentreadily available source for mesenchymal stem cells. Moreover,mesenchymal stem cells can be derivable from adipose tissue and bonemarrow stromal cells and speculated to be present in other tissues.While there are dramatic qualitative and quantitative differences in theorgans from which adult stem cells can be derived, the initialdifferences between the cells may be relatively superficial and balancedby the similar range of plasticity they exhibit.

Homogeneous human mesenchymal stem cell compositions are provided whichserve as the progenitors for all mesenchymal cell lineages. MSCs areidentified by specific cell surface markers which are identified withunique monoclonal antibodies. The homogeneous MSC compositions areobtained by positive selection of adherent marrow or periosteal cellswhich are free of markers associated with either hematopoietic cell ordifferentiated mesenchymal cells. These isolated mesenchymal cellpopulations display epitopic characteristics associated with onlymesenchymal stem cells, have the ability to regenerate in culturewithout differentiating, and have the ability to differentiate intospecific mesenchymal lineages when either induced in vitro or placed invivo at the site of damaged tissue.

In order to obtain subject human mesenchymal stem cells, pluripotentmesenchymal stem cells are separated from other cells in the bone marrowor other MSC source. Bone marrow cells may be obtained from iliac crest,femora, tibiae, spine, rib or other medullary spaces. Other sources ofhuman mesenchymal stem cells include embryonic yolk sac, placenta,umbilical cord, fetal and adolescent skin, and blood.

As discussed above, the mesenchymal stem cells can be isolated andpurified by different methods. A preferred method has been described indetail in the examples section which follows. Other methods include,providing a tissue specimen containing mesenchymal stem cells, addingcells from the tissue specimen to a medium which contains factors thatstimulate mesenchymal stem cell growth without differentiation andallows, when cultured, for the selective adherence of only themesenchymal stem cells to a substrate surface, culturing thespecimen-medium mixture, and removing the non-adherent matter from thesubstrate surface.

In a preferred embodiment, the mesenchymal stem cells are derived fromone or sources comprising: autologous, heterologous, syngeneic,allogeneic or xenogeneic sources. These sources can include cell lines.As used herein, “source” refers to the animal in which these stem cellswere obtained from, including human.

Differentiation of mesenchymal stem cells to the cardiac lineage iscontrolled by factors present in the cardiac environment. Localchemical, electrical and mechanical environmental influences alterpluripotent MSCs and convert the cells administered to the heart intothe cardiac lineage.

In a preferred embodiment, mesenchymal stem cells differentiate intolineages of cells that make up the different heart tissues. In someembodiments, the lineages are identified by cardiac cell specificmarkers comprising cardiac transcription factor GATA-4, MDR1;endothelial cell markers Factor VIII and KDR; vascular smooth musclemarker α-smooth muscle actin; or cardiomyocyte marker α-sarcomericactinin. For example, detection of expression of cardiomyocyte specificproteins is achieved using antibodies to, for example, myosin heavychain monoclonal antibody MF 20 (MF20), sarcoplasmic reticulum calciumATPase (SERCA1) (mnAb 10D1) or gap junctions using antibodies toconnexin 43. Other markers for cardiomyocytes comprise cardiac troponinI (cTnT), cardiac troponin T (cTnT), sarcomeric myosin heavy chain(MHC), GATA-4, Nkx2.5, N-cadherin, β1-adrenoceptor β1-AR), ANF, theMEF-2 family of transcription factors, creatine kinase MB (CK-MB),myoglobin, or atrial natriuretic factor (ANF).

GATA transcription factor includes members of the GATA family of zincfinger transcription factors. GATA transcription factors are involved inthe development of several mesodermally derived cell lineages.Preferably, GATA transcription factors include GATA-4 and/or GATA-6. TheGATA-6 and GATA-4 proteins share high-level amino acid sequence identityover a proline-rich region at the amino terminus of the protein that isnot conserved in other GATA family members.

In another preferred embodiment, soluble factors from mesenchymal stemcell cultures are administered to a patient with heart disease ordisorders thereof. The factors are administered in an effective amountresulting in the localization, proliferation and maturation of cardiacstem cells into cells of the damaged heart tissue.

In another preferred embodiment, the mesenchymal cells are administeredto a patient, for treating heart disease or disorders thereof. The cellscan be autologous or donor derived. The administration of themesenchymal stem cells results in the recruitment of cardiac stem cellsto the damaged tissues, accelerated proliferation and differentiation ofthe cardiac stem cells and the repair of the damaged tissues. Inpreferred embodiments, the cardiac stem cells are endogenous stem cells.In other embodiments, the cardiac stem cells can be donor derived. Thecombinations of origins of the mesenchymal stem cells and cardiac stemcells can be in any combinations. For example, mesenchymal stem cellsare autologous and the cardiac stem cells are endogenous. In otheralternatives the mesenchymal stem cells are donor derived and thecardiac stem cells are endogenous. In another embodiment, themesenchymal stem cells are autologous and the cardiac stem cells aredonor derived. In other embodiments the mesenchymal stem cells compriseboth autologous and donor derived cells and the cardiac stem cellscomprise donor derived and endogenous cells. In other embodiments,soluble factors from mesenchymal stem cells are administered to thedamaged heart tissue for recruiting, accelerating proliferation anddifferentiation of cardiac stem cells.

In a preferred embodiment, cardiac stem cells are identified by markerscomprising: c-kit^(pos), CD3^(neg), CD14^(neg) and CD68^(neg).

Cardiac injury promotes tissue responses which enhance myogenesis usingimplanted MSCs. Thus, MSCs are introduced to the infarct zone to reducethe degree of scar formation and to augment ventricular function. Newmuscle is thereby created within an infarcted myocardial segment. MSCsare directly infiltrated into the zone of infarcted tissue. Theintegration and subsequent differentiation of these cells ischaracterized, as described above. Timing of intervention is designed tomimic the clinical setting where patients with acute myocardialinfarction would first come to medical attention, receive first-linetherapy, followed by stabilization, and then intervention withmyocardial replacement therapy if necessary.

Of the four chambers of the heart, the left ventricle is primarilyresponsible for pumping blood under pressure through the body'scirculatory system. It has the thickest myocardial walls and is the mostfrequent site of myocardial injury resulting from congestive heartfailure. The degree of advance or severity of the congestive heartfailure ranges from those cases where heart transplantation is indicatedas soon as a suitable donor organ becomes available to those wherelittle or no permanent injury is observed and treatment is primarilyprophylactic,

The severity of resulting myocardial infarction, i.e. the percentage ofmuscle mass of the left ventricle that is involved can range from about1 to about 80 percent. This represents affected tissue areas, whether asone contiguous ischemia or the sum of smaller ischemic lesions, havinghorizontal affected areas from about 1 cm² to about 6 cm² and athickness of from 1-2 mm to 1-1.5 cm. The severity of the infarction issignificantly affected by which vessel(s) is involved and how much timehas passed before treatment intervention is begun.

The mesenchymal stem cells used in accordance with the invention are, inorder of preference, autologous, allogeneic or xenogeneic, and thechoice can largely depend on the urgency of the need for treatment. Apatient presenting an imminently life threatening condition may bemaintained on a heart/lung machine while sufficient numbers ofautologous MSCs are cultured or initial treatment can be provided usingother than autologous MSCs.

The MSC therapy of the invention can be provided by several routes ofadministration, including the following. First, intracardiac muscleinjection, which avoids the need for an open surgical procedure, can beused where the MSCs are in an injectable liquid suspension preparationor where they are in a biocompatible medium which is injectable inliquid form and becomes semi-solid at the site of damaged myocardium. Aconventional intracardiac syringe or a controllable arthroscopicdelivery device can be used so long as the needle lumen or bore is ofsufficient diameter (e.g. 30 gauge or larger) that shear forces will notdamage the MSCs. The injectable liquid suspension MSC preparations canalso be administered intravenously, either by continuous drip or as abolus. During, open surgical procedures, involving direct physicalaccess to the heart, all of the described forms of MSC deliverypreparations are available options.

As a representative example of a dose range is a volume of about 20 toabout 50 μl of injectable suspension containing about 10-40×10⁶ MSCs/ml.The concentration of cells per unit volume, whether the carrier mediumis liquid or solid remains within substantially the same range. Theamount of MSCs delivered will usually be greater when a solid, “patch”type application is made during an open procedure, but follow-up therapyby injection will be as described above. The frequency and duration oftherapy will, however, vary depending on the degree (percentage) oftissue involvement, as already described (e.g. 5-40% left ventricularmass).

In cases having in the 5-10% range of tissue involvement, it is possibleto treat with as little as a single administration of one million MSCsin 20-50 μl of injection preparation. The injection medium can be anypharmaceutically acceptable isotonic liquid. Examples include phosphatebuffered saline (PBS), culture media such as DMEM (preferablyserum-free), physiological saline or 5% dextrose in water.

In cases having more in a range around the 20% tissue involvementseverity level, multiple injections of 20-50 μl (10-40×10⁶ MSCs/nil) areenvisioned. Follow-up therapy may involve additional dosings.

In very severe cases, e.g. in a range around the 40% tissue involvementseverity level, multiple equivalent doses for a more extended durationwith long term (up to several months) maintenance dose aftercare maywell be indicated.

In another embodiment, the isolated and culture expanded mesenchymalstem cells can be utilized for the implantation of various prostheticdevices. For example, using porous ceramic structures filled withculture-expanded human mesenchymal stem cells, and implanting thesestructures in areas where there is extensive tissue damage.

Additional Types of Stem Cells

In other embodiments, other stem cells can be used with the mesenchymalstem cells in the treatment of heart diseases and disorders thereof.Preferably, the stem cells are totipotent or pluripotent stem cells.

There are many undifferentiated cells found in vivo. Stem cells areundifferentiated immature cells, capable of self renewal (divisionwithout limit) and differentiation (specialization). These juvenilecells are abundant in a developing embryo; however, their numbersdecrease as development progresses. By contrast, an adult organismcontains limited number of stem cells which are confined to certain bodycompartments.

It is generally believed that stem cells are either: monopotent,bipotent or pluripotent. Monopotent and bipotent stem cells are morerestricted in development and give rise to one or two types ofspecialized cells, respectively. In contrast, the pluripotent stem cells(PSCs) can differentiate into many different types of cells, giving riseto tissue (which constitute organs) or in the case of totipotent stemcells, the whole organism. Pluripotent stem cells, unlike monopotent orbipotent, are capable of multilineage differentiation, giving rise to atissue which would consist of a collection of cells of different typesor lineages.

According to the current understanding, a stem cell, such as apluripotent stem cell, has the following four characteristics: (i) it isan undifferentiated cell—i.e. it is not terminally differentiated; (ii)it has the ability to divide without limit; (iii) it has the ability togive rise to differentiated progeny; and (iv) when it divides eachdaughter has a choice: it can either remain as stem cell like its parentor it can embark on a course leading to differentiation.

The hematopoietic stem cell is an example of a pluripotent stem cellwhich is found among marrow cells and gives rise to all the variousblood cells (including leucocytes and erythrocytes). Hemopoietic stemcells can be extracted by isolation from (i) bone marrow, (ii) growthfactor mobilized peripheral blood or (iii) cord blood (placenta).Recently, hemopoietic stem cells have been prepared from Embryonic Stemcells (ES), which are extracted from embryos obtained using in vitrofertilization techniques. These undifferentiated cells are capable ofmulti-lineage differentiation and reconstitution of all body tissue i.e.are totipotent.

There are a number of undifferentiated stem cells of the hemopoieticlineage. These include pluripotent stem cells (PSCs), lymphoid stemcells (LSCs) and myeloid stem cells known collectively aslymphohaematopoietic progenitor cells (LPCs). LSCs and myeloid stemcells are each formed by the differentiation of PSCs. Hence, LSCs andmyeloid stem cells are more committed than PSCs. Examples ofdifferentiated cells of the hemopoietic lineage include T cells, Bcells, eosinophils, basophils, neutrophils, megakaryocytes, monocytes,granulocytes, mast cells, and lymphocytes.

Other stem cells include neural stem cells, multipotent stem cells thatcan generate neurons, atrocytes and oligodendrocytes (Nakafuku andNakamura, 1995, J. Neurosci Res., vol 41(2): 153-68; Anderson, 1994,FASEB J., vol 8(10); 707-13; Morshead of al., 1994, Neuron, Vol 13(5):1071-82). Skeletal muscle satellite cells are another type of stem cell,more specifically a distinct class of myogenic cells that are maintainedas quiescent stem cells in the adult and can give rise to new musclecells when needed (Bischoff, 1986, Dev Biol., vol 115(1): 129-39). Othertypes of stem cells are epithelial stem cells, a subset of basal cells,and mesenchymal stem cells.

Embryonic stem (ES) cells are routinely used in the production oftransgenic animals. ES cells have been shown to differentiate in vitrointo several cell types including lymphoid precursors (Potocnik et al.,1994, EMBO J., vol 13(22): 5274-83) and neural cells. ES cells arecharacterized by a number of stage-specific markers such asstage-specific embryonic markers 3 and 4 (SSEA-3 and SSEA-4), highmolecular weight glycoproteins TRA-1-60 and TRA-1-81 and alkalinephosphatase (Andrews et al., 1984, Hybridoma, vol 3: 347-361; Kannagi etal., 1983, EMBO J., vol 2: 2355-2361; Fox et al., 1984, Dev. Biol., vol103: 263-266; Ozawa et al., 1985, Cell. Differ., vol 16: 169-173).

Various antigens are associated with undifferentiated and differentiatedcells. The term “associated” here means the cells expressing or capableof expressing, or presenting or capable of being induced to present, orcomprising, the respective antigen(s). Most undifferentiated cells anddifferentiated cells comprise Major Histocompatibility Complex (MHC)Class I antigens and/or Class II antigens. If these antigens areassociated with those cells then they are called Class I⁺ and/or ClassII⁺ cells. Each specific antigen associated with an undifferentiatedcell or a differentiated cell can act as a marker. Hence, differenttypes of cells can be distinguished from each other on the basis oftheir associated particular antigen(s) or on the basis of a particularcombination of associated antigens. Examples of these marker antigensinclude the antigens CD34, CD19 and CD3. If these antigens are presentthen these particular cells are called CD34⁺, CD19⁺ and CD3⁺ cellsrespectively. If these antigens are not present then these cells arecalled CD34⁻, CD19⁻ and CD3⁻ cells respectively.

Some of the markers identified on myeloid stem cells comprise CD34⁺ DR⁺,CD13⁺, CD33⁺, CD7⁺ and TdT⁺ cells. PSCs are CD34⁺ DR⁻TdT⁻ cells (otheruseful markers being CD38⁻ and CD36⁺). LSCs are DR⁺, CD34⁺ and TdT⁺cells (also CD38⁺). Embryonic stem cells express SSEA-3 and SSEA-4, highmolecular weight glycoproteins TRA-1-60 and TRA-1-81 and alkalinephosphatase. They also do not express SSEA-1, the presence of which isan indicator of differentiation. Other markers are known for other typesof stem cells, such as Nestein for neuroepithelial stem cells (J.Neurosci, 1985, Vol 5: 3310). Mesenchymal stem cells are also positivefor SH2, SH3, CD29, CD44, CD7I, CD90, CD106, CD120a and CD124, forexample, and negative for CD34, CD45 and CD14.

Stem cells may further be isolated for transduction and differentiationusing known methods. For example, in mice, bone marrow cells areisolated by sacrificing the mouse and cutting the leg bones with a pairof scissors. Stem cells may also be isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4⁺ and CD8+ (T cells), CD45⁺ (panB cells), GR-1(granulocytes), and lad (differentiated antigen presenting cells). Foran example of this protocol see, Inaba et al., J. Exp. Med.176:1693-1702 (1992).

In humans, CD34⁺ hematopoietic stem cells can be obtained from a varietyof sources including cord blood, bone marrow, and mobilized peripheralblood. Purification of CD34⁺ cells can be accomplished by antibodyaffinity procedures. An affinity column isolation procedure forisolating CD34⁺ cells is described by Ho et al., Stem Cells 13 (suppl.3): 100-105 (1995). See also, Brenner, Journal of Hematotherapy 2: 7-17(1993). Methods for isolating, purifying and culturally expandingmesenchymal stem cells are known.

Alternatively, or in addition, many cells can be identified bymorphological characteristics. The identification of cells usingmicroscopy, optionally with staining techniques is an extremely welldeveloped branch of science termed histology and the relevant skills arewidely possessed in the art.

Various techniques may be employed to separate the cells by initiallyremoving cells of dedicated lineage. Monoclonal antibodies areparticularly useful for identifying markers associated with particularcell lineages and/or stages of differentiation.

If desired, a large proportion of terminally differentiated cells may beremoved by initially using a “relatively crude” separation. For example,magnetic bead separations may be used initially to remove large numbersof lineage committed cells. Desirably, at least about 80%, usually atleast 70% of the total hematopoietic cells will be removed.

Procedures for separation may include but are not limited to, magneticseparation, using antibody-coated magnetic beads, affinitychromatography, cytotoxic agents joined to a monoclonal antibody or usedin conjunction with a monoclonal antibody, including but not limited to,complement and cytotoxins, and “panning” with antibody attached to asolid matrix, e.g., plate, elutriation or any other convenienttechnique.

Techniques providing accurate separation include but are not limited to,flow cytometry, which can have varying degrees of sophistication, e.g.,a plurality of color channels, low angle and obtuse light scatteringdetecting channels, impedance channels, etc.

Cardiotropic Agents:

In some aspects of the invention, one or more “cardiotropic factors” canbe included in the culture medium if it is desired by a user todifferentiate the stem cells. These are factors that either alone or incombination enhance proliferation or survival of cardiomyocyte typecells, or inhibit the growth of other cell types. The effect may be dueto a direct effect on the cell itself, or due to an effect on anothercell type, which in turn enhances cardiomyocyte formation. For example,factors that induce the formation of hypoblast or epiblast equivalentcells, or cause these cells to produce their own cardiac promotingelements, all come within the rubric of cardiotropic factors.

Factors thought to induce differentiation of pluripotent stem cells intocells of the mesoderm layer, or facilitate further differentiation intocardiomyocyte lineage cells include the following: Nucleotide analogsthat affect DNA methylation and altering expression ofcardiomyocyte-related genes TGF-β ligands (exemplified by TGF-β1,TGF-β2, TGF-β3 and other members of the TGF-β superfamily). Ligands binda TGF-β receptor activate Type I and Type II serine kinases and causephosphorylation of the Smad effector. Morphogens like Activin A andActivin B (members of the TGF-β superfamily); Insulin-like growthfactors (such as IGF II); Bone morphogenic proteins (members of theTGF-β superfamily, exemplified by BMP-2 and BMP-4); Fibroblast growthfactors (exemplified by bFGF, FGF-4, and FGF-8) and other ligands thatactivate cytosolic kinase raf-1 and mitogen-activated proteins kinase(MAPK); Platelet-derived growth factor (exemplified by PDGFβ)Natriuretic factors (exemplified by atrial natriuretic factor (ANF),brain natriuretic peptide (BNP). Related factors such as insulin,leukemia inhibitory factor (LIF), epidermal growth factor (EGF), TGFα,and products of the cripto gene. Specific antibodies with agonistactivity for the same receptors. Alternatively or in addition, the cellscart be cocultured with cells (such as endothelial cells of variouskinds) that secrete factors enhancing cardiomyocyte differentiation.Nucleotide analogs that affect DNA methylation (and thereby influencegene expression) can effectively be used to increase the proportion ofcardiomyocyte lineage cells that emerge following initialdifferentiation. For example, it has been found that inclusion of5-aza-deoxy-cytidine in the culture medium increases the frequency ofcontracting cells in the population, and expression of cardiac αMHC.

Particularly effective combinations of cardiotropic agents include useof a morphogen like Activin A and a plurality of growth factors, such asthose included in the TGF-β and IGF families during the early commitmentstage, optionally supplemented with additional cardiotropins such as oneor more fibroblast growth factors, bone morphogenic proteins, andplatelet-derived growth factors.

During the elaboration of this invention, it was found that omittingfactors such as insulin-like growth factor II (IGF II) and relatedmolecules from the final stages of in vitro differentiation actuallyincreased the levels of cardiac gene expression. In unrelated studies,IGF II has been found to decrease the levels of GSK3β in fibroblasts(Scalia et al, J. Cell. Biochem. 82:610, 2001). IGF II may thereforepotentiate the effects of Wnt proteins present in the culture medium orsecreted by the cells. Wnt proteins normally stabilize and cause nucleartranslocation of a cytoplasmic molecule, β-catenin, which comprises aportion of the transcription factor TCF. This changes transcriptionalactivity of multiple genes. In the absence of Wnt, β-catenin isphosphorylated by the kinase GSK3β, which both destabilizes β-cateninand keeps it in the cytoplasm.

Since Wnt activators like IL-2 apparently limit cardiomyocytedifferentiation, it is believed that culturing with Wnt antagonists canincrease the extent or proportion of cardiomyocyte differentiation ofhES cells. Wnt signaling can be inhibited by injection of synthetic mRNAencoding either DKK-1 or Crescent (secreted proteins that bind andinactivate Wnts) (Schneider et al., Genes Dev. 15:304, 2001), or byinfection with a retrovirus encoding DKK-1 (Marvin et al., Genes Dev.15:316, 2001). Alternatively, the Wnt pathway can be inhibited byincreasing the activity of the kinase GSK3β, for example, by culturingthe cells with factors such as IL-6 or glucocorticoids.

The combinations and amounts of such compounds that are effective forenriching cardiomyocyte production can be determined empirically byculturing undifferentiated or early differentiated embryonic stem cellsor their progeny in a culture environment incorporating such factors,and then determining whether the compound has increased the number ofcardiomyocyte lineage cells in the population according to thephenotypic markers listed below.

Following initial differentiation (and before or after a separationstep, if employed), it is possible to increase the percentage ofcardiomyocyte lineage cells by culturing in an environment containing a“cardiomyocyte enrichment agent”. This is a factor in the medium or on asurface substrate that promotes the outgrowth of the desired celltype—either by facilitating proliferation of cardiomyocyte lineagecells, or by inhibiting the growth (or causing apoptosis) of cells ofother tissue types. Some of the cardiotropic factors listed above aresuitable for this purpose. Also suitable are certain compounds knownbeneficial to cardiomyocytes in vivo, or their analogs. Included arecompounds capable of forming a high energy phosphate bond (such ascreatine); an acyl group carrier molecule (such as carnitine); and acardiomyocyte calcium channel modulator (such as taurine).

Cardiomyocyte specific markers comprise: Cardiac troponin I (cTnI), asubunit of troponin complex that provides a calcium-sensitive molecularswitch for the regulation of striated muscle contraction. Cardiactroponin T (cTnT) Atrial natriuretic factor (ANF), hormone expressed indeveloping heart and fetal cardiomyocytes but down-regulated in adults.It is considered a good marker for cardiomyocytes because it isexpressed in a highly specific manner in cardiac cells but not skeletalmyocytes. The cells will also typically express at least one (and oftenat least 3, 5, or more) of the following markers: sarcomeric myosinheavy chain (MHC) Titin, tropomyosin, α-actinin, and desmin, GATA-4, atranscription factor that is highly expressed in cardiac mesoderm andpersists in the developing heart. It regulates many cardiac genes andplays a role in cardiogenesis Nkx2.5, a cardiac transcription factorexpressed in cardiac mesoderm during early mouse embryonic development,which persists in the developing heart. MEF-2A, MEF-2B, MEF-2C, MEF-2D;transcription factors that are expressed in cardiac mesoderm and persistin developing heart N-cadherin, which mediates adhesion among cardiaccells Connexin 43, which forms the gap junction between cardiomyocytes.β1-adrenoceptor (β1-AR) creatine kinase MB (CK-MB) and myoglobin, whichare elevated in serum following myocardial infarction. Other markersthat may be positive on cardiomyocytes and their precursors includeα-cardiac actin, early growth response-I, and cyclin D2.

Tissue-specific markers can be detected using any suitable immunologicaltechnique—such as flow immunocytochemistry or affinity adsorption forcell-surface markers, immunocytochemistry (for example, of fixed cellsor tissue sections) for intracellular or cell-surface markers, Westernblot analysis of cellular extracts, and enzyme-linked immunoassay, forcellular extracts or products secreted into the medium. Expression of anantigen by a cell is said to be antibody-detectable if a significantlydetectable amount of antibody will bind to the antigen in a standardimmunocytochemistry or flow cytometry assay, optionally after fixationof the cells, and optionally using a labeled secondary antibody or otherconjugate (such as a biotin-avidin conjugate) to amplify labeling.

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods. See U.S. Pat. No. 5,843,780 for details ofgeneral technique. Sequence data for other markers listed in thisdisclosure can be obtained from public databases such as GenBank (URLwww.ncbi.nlm.nlh.gov:80/entrez). Expression at the mRNA level is said tobe detectable according to one of the assays described in thisdisclosure if the performance of the assay on cell samples according tostandard procedures in atypical controlled experiment results in clearlydiscernable hybridization or amplification product. Expression oftissue-specific markers as detected at the protein or mRNA level isconsidered positive if the level is at least 2-fold, and preferably morethan 10- or 50-fold above that of a control cell, such as anundifferentiated pluripotent stem cell or other unrelated cell type.

Once markers have been identified on the surface of cells of the desiredphenotype, they can be used for immunoselection to further enrich thepopulation by techniques such as immunopanning or antibody-medicatedfluorescence-activated cell sorting.

Gene Therapy

In some preferred embodiments, the mesenchymal stem cells comprisetherapeutic genes for delivery into the body, such as for example, theheart. In some embodiments, the therapeutic gene is a transgene. Forexample, the delivery cells—e.g. the mesenchymal stem cells are removedfrom the body, and a therapeutic transgene is introduced into them viavehicles well known to those skilled in the art such as those used indirect-gene-transfer methods. For example, while still in thelaboratory, the genetically modified cells are tested and then allowedto grow and multiply and, finally, are infused back into the patient.Alternatively, allogeneic cells that bear normal, endogenous genes canreverse a deficiency in a particular target tissue. Use of cells bearingeither transgenes or normal, endogenous genes is referred to herein asgene therapy.

Gene therapy using genetically modified cells offers several uniqueadvantages over direct gene transfer into the body. First the additionof the therapeutic transgene to the delivery cells takes place outsidethe patient, which allows the clinician an important measure of controlbecause they can select and work only with those cells that both containthe transgene and produce the therapeutic agent in sufficient quantity.

In some embodiments, the mesenchymal stem cells express stem cellrecruiting factors, growth factors, therapeutic factors, endogenousfactors such as, for example, cardiac troponin I (cTnI), cardiactroponin T (cTnT), atrial natriuretic factor (ANF), and the like. Inview of the foregoing, the methods according to the present inventionare useful for targeting a gene of interest (either a transgene or anendogenous gene) to a tissue in a mammal by introducing a cellcomprising the gene of interest to the mammal. Such methods are usefulfor treating a disease characterized by a deficiency in a gene productin a mammal by administering a cell comprising a functional geneencoding the gene product into the mammal and administering aglycoconjugate to the mammal. Stem cells may be used as a vehicle fordelivering genes to specific tissues in the body. Stem cell-basedtherapies are a major area of investigation in cancer research.

Embodiments of the invention further provide localizing of transfusedcells such as stem cells to provide a functional gene to a patientsuffering from a disease caused by a lack of that gene, or, recruitmentof endogenous stem cells to the damaged heart tissue. By providing agene that allows for recruitment, stimulation and proliferation ofcardiac stem cells, the treatment of heart disease or disorders thereofcan be accelerated even further. Therefore, the present invention alsoprovides the ability to direct the localization of the transfused cellssuch as allogeneic stem cells that have a stable, normal gene. Suchtransfused cells then create a stable micro-chimera of the recipient.

Recruiting the stem cells to the target site can be induced artificiallyby administering a suitable chemokine systemically or at the desiredsite via injection or through expression from the mesenchymal stemcells. A suitable molecule is hypoxia inducible factor-1, a chemokinesuch as stromal derived factor-1 (SDF-1). Endothelial stem cells mayalso be recruited to the desired site by means of an interleukin, suchas IL-1 or IL-8.

It may be desirable that the cells have the ability to replicate incertain drug screening and therapeutic applications, and to provide areservoir for the generation of cardiomyocytes and their precursors. Thecells can optionally be telomerized to increase their replicationpotential, either before or after they progress to restricteddevelopmental lineage cells or terminally differentiated cells. Stemcells that are telomerized may be taken down the differentiation pathwaydescribed earlier; or differentiated cells can be telomerized directly.

Cells are telomerized by genetically altering them by transfection ortransduction with a suitable vector, homologous recombination, or otherappropriate technique, so that they express the telomerase catalyticcomponent (TERT), typically under a heterologous promoter that increasestelomerase expression beyond what occurs under the endogenous promoter.Particularly suitable is the catalytic component of human telomerase(hTERT), provided in International Patent Application WO 98/14592. Forcertain applications, species homologs like mouse TERT (WO99/27113) canalso be used. Transfection and expression of telomerase in human cellsis described in Bodnar et al., Science 279:349, 1998 and Jiang et al.,Nat. Genet. 21:111, 1999. In another example, hTERT clones (WO 98/14592)are used as a source of hTERT encoding sequence, and spliced into anEcoRI site of a PBBS212 vector under control of the MPSV promoter, orinto the EcoRI site of commercially available pBABE retrovirus vector,under control of the LTR promoter.

Differentiated or undifferentiated stem cells are genetically alteredusing vector containing supernatants over a 8-16 h period, and thenexchanged into growth medium for 1-2 days. Genetically altered cells areselected using a drug selection agent such as puromycin, G418, orblasticidin, and then recultured. They can then be assessed for hTERTexpression by RT-PCR, telomerase activity (TRAP assay),immunocytochemical staining for hTERT, or replicative capacity. Thefollowing assay kits are available commercially for research purposes:TRAPeze™, XL Telomerase Detection Kit (Cat. s7707; Intergen Co.,Purchase N.Y.); and TeloTAGGG Telomerase PCR ELISAplus (Cat. 2,013,89;Roche Diagnostics, Indianapolis Ind.). TERT expression can also beevaluated at the mRNA by RT-PCR. Available commercially for researchpurposes is the LightCycler TeloTAGGG hTERT quantification kit (Cat.3,012,344; Roche Diagnostics). Continuously replicating colonies areenriched by further culturing under conditions that supportproliferation, and cells with desirable phenotypes can optionally becloned by limiting dilution.

In certain embodiments, stem cells are differentiated into cardiomyocyteprecursors, and then genetically altered to express TERT. In otherembodiments, stem cells are genetically altered to express TERT, andthen differentiated into cardiomyocyte precursors or terminallydifferentiated cells. Successful modification to increase TERTexpression can be determined by TRAP assay, or by determining whetherthe replicative capacity of the cells has improved.

Depending on the intended use of the cells, other methods ofimmortalization may also be acceptable, such as transforming the cellswith DNA encoding mye, the SV40 large T antigen, or MOT-2 (U.S. Pat. No.5,869,243, International Patent Applications WO 97/32972 and WO01/23555). Transfection with oncogenes or oncovirus products is lesssuitable when the cells are to be used for therapeutic purposes.Telomerized cells are of particular interest in applications of where itis advantageous to have cells that can proliferate and maintain theirkaryotype—for example, in pharmaceutical screening, and in therapeuticprotocols where differentiated cells are administered to an individualin order to augment cardiac function.

The cells can also be genetically altered in order to enhance theirability to be involved in tissue regeneration, or to deliver atherapeutic gene to a site of administration. A vector is designed usingthe known encoding sequence for the desired gene, operatively linked toa promoter that is either pan-specific or specifically active in thedifferentiated cell type. Of particular interest are cells that aregenetically altered to express one or more growth factors of varioustypes, cardiotropic factors such as atrial natriuretic factor, cripto,and cardiac transcription regulation factors, such as GATA-4, Nkx2.5,and MEF2-C. Production of these factors at the site of administrationmay facilitate adoption of the functional phenotype enhance thebeneficial effect of the administered cell, or increase proliferation oractivity of host cells neighboring the treatment site.

Introducing Transgenes Into Stem Cells

Means for introducing transgenes into cells are well known. A variety ofmethods for delivering and expressing a nucleic acid within a mammaliancell are known to those of ordinary skill in the art. Such methodsinclude, for example viral vectors, liposome-based gene delivery (WO93/24640; Mannino Gould-Fogerite, BioTechniques 6(7):682-691 (1988);U.S. Pat. No. 5,279,833; WO 91/06309; Feigner et at, Proc. Natl. Acad.Sci. USA 84:7413-7414 (1987); and Budker et al., Nature Biotechnology,14(6):760-764 (1996)). Other methods known to the skilled artisaninclude electroporation (U.S. Pat. Nos. 5,545,130, 4,970,154, 5,098,843,and 5,128,257), direct gene transfer, cell fusion, precipitationmethods, particle bombardment, and receptor-mediated uptake (U.S. Pat.Nos. 5,547,932, 5,525,503, 5,547,932, and 5,460,831). See also, U.S.Pat. No. 5,399,346.

Widely used retroviral vectors include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immune deficiencyvirus (SW), human immuno deficiency virus (HIV), and combinationsthereof. See, e.g., Buchscher et at, J. Virol. 66(5):2731-2739 (1992);Johann et al., J. Virol. 66(5):1635-1640 (1992); Sommerfelt et al.,Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989);Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700, andRosenburg & Fauci, in Fundamental Immunology, Third Edition (Paul ed.,1993)).

AAV-based vectors are also used to transduce cells with target nucleicacids, e.g., in the in vitro production of nucleic acids andpolypeptides, and in vivo and ex vivo gene therapy procedures. See, Westet al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641;Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invst.94:1351 (1994) and Samulski (supra) for an overview of AAV vectors.Construction of recombinant AAV vectors are described in a number ofpublications, including Lebkowski, U.S. Pat. No. 5,173,414; Tratschin etal., Mol. Cell. Biol. 5(11):3251-3260 (1985); Tratschin et al., Mol.Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, Proc. Natl. Acad.Sci. USA 81:6466-6470 (1984); and Samulski et al., J Virol.63:03822-3828 (1989).

Retroviral vectors are typically used for cells useful in the presentinvention. Such vectors may comprise, for example, an HIV-2 packageablenucleic acid packaged in an HIV-2 particle, typically using a packagingcell line. Cell transduction vectors have considerable commercialutility as a method of introducing genes into target cells. Inparticular, gene therapy procedures, in which the cell transductionvectors of the invention are used to transduce target cells with atherapeutic nucleic acid in an in vivo or ex vivo procedure may be used.

Stem cells such as CD34⁺ stem cells may be used in ex vivo proceduresfor cell transduction and gene therapy. The present invention utilizesthe feature that stem cells differentiate into other cell types invitro, or can be introduced into a mammal (such as the donor of thecells) where they will engraft in the heart for differentiation. Hence,embodiments extend to directing stem cells to particular organs toregenerate tissue, such as to the heart to regenerate cardiac musclecells. Stem cells, such as, for example, pluripotent stem cells, canalso be used in conjunction with the mesenchymal stem cells. If it isdesired, the stem cells can also be differentiated in vitro. Methods fordifferentiating CD34⁺ cells in vitro into clinically important immunecell types using cytokines such a GM-CSF, IFNγ and TNFα are known (See,Inaba et al, J. Exp. Med. 176, 1693-1702 (1992)). Yu et al., PNAS 92:699-703 (1995) describe a method of transducing CD34⁺ cells from humanfetal cord blood using retroviral vectors.

Drug Screening

Cells of this invention can be used to screen for factors (such assolvents, small molecule drugs, peptides, oligonucleotides) orenvironmental conditions (such as culture conditions or manipulation)that affect the characteristics of such cells and their various progeny.

In some applications, mesenchymal stem cells or other stem cell typesare used to screen factors that promote maturation into later-stagecardiomyocyte precursors, or terminally differentiated cells, or topromote proliferation and maintenance of such cells in long-termculture. For example, candidate maturation factors or growth factors aretested by adding them to cells in different wells, and then determiningany phenotypic change that results, according to desirable criteria forfurther culture and use of the cells.

Other screening applications of this invention relate to the testing ofpharmaceutical compounds for their effect on cardiac muscle tissuemaintenance or repair. Screening may be done either because the compoundis designed to have a pharmacological effect on the cells, or because acompound designed to have effects elsewhere may have unintended sideeffects on cells of this tissue type. The screening can be conductedusing any of the stem cells or terminally differentiated cells.

The reader is referred generally to the standard textbook In vitroMethods in Pharmaceutical Research, Academic Press, 1997, and U.S. Pat.No. 5,030,015. Assessment of the activity of candidate pharmaceuticalcompounds generally involves combining the cells of this invention withthe candidate compound, either alone or in combination with other drugs.The investigator determines any change in the morphology, markerphenotype, or functional activity of the cells that is attributable tothe compound (compared with untreated cells or cells treated with aninert compound), and then correlates the effect of the compound with theobserved change.

Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and the expression of certainmarkers and receptors. Effects of a drug on chromosomal DNA can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. The reader isreferred to A. Vickers (pp 375-410 in In vitro Methods in PharmaceuticalResearch, Academic Press, 1997) for further elaboration.

Effect of cell function can be assessed using any standard assay toobserve phenotype or activity of cardiomyocytes, such as markerexpression, receptor binding, contractile activity, orelectrophysiology—either in cell culture or in vivo. Pharmaceuticalcandidates can also be tested for their effect on contractileactivity—such as whether they increase or decrease the extent orfrequency of contraction. Where an effect is observed, the concentrationof the compound can be titrated to determine the median effective dose(ED₅₀).

Treatment

The amount of stem cells administered to the patient will also varydepending on the condition of the patient and should be determined viaconsideration of all appropriate factors by the practitioner.Preferably, however, about 1×10⁶ to about 1×10¹², more preferably about1×10⁸ to about 1×10¹¹, more preferably, about 1×10⁹ to about 1×10¹⁰ stemcells are utilized for adult humans. These amounts will vary dependingon the age, weight, size, condition, sex of the patient, the type oftumor to be treated, the route of administration, whether the treatmentis regional or systemic, and other factors. Those skilled in the artshould be readily able to derive appropriate dosages and schedules ofadministration to suit the specific circumstance and needs of thepatient.

Methods of re-introducing cellular components are known in the art andinclude procedures such as those exemplified in U.S. Pat. No. 4,844,893to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg.

Pharmaceutical Compositions

In other embodiments, the present invention provides pharmaceuticalcompositions comprising mesenchymal stem cells. In other preferredembodiments, pharmaceutical compositions comprise a mesenchymal stemcell and embryonic stem cells.

In other aspects, the present invention features kits for treatingcardiac tissue damage or for delivering a functional gene or geneproduct to the heart in a mammal comprising a mesenchymal stem cell.Stem cells generally have been presented to the desired organs either byinjection into the tissue, by infusion into the local circulation, or bymobilization of autologous stem cells from the marrow accompanied byprior removal of stem cell-entrapping organs before mobilization whenfeasible, i.e., splenectomy.

In some embodiments, the administration of the stem cell compositionscan be coupled with other therapies. For example, a therapeutic agentcan be administered prior to, concomitantly with, or after infusing thestem cells to a patient.

Administration of cells transduced ex vivo can be by any of the routesnormally used for introducing a cell or molecule into ultimate contactwith blood or tissue cells. The stem cells may be administered in anysuitable manner, preferably with pharmaceutically acceptable carriers.Suitable methods of administering such cells in the context of thepresent invention to a patient are available, and, although more thanone route can be used to administer a particular composition, aparticular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.Parenteral administration is one useful method of administration. Theformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and in some embodiments, can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water, forinjections, immediately prior to use. These formulations may beadministered with factors that mobilize the desired class of adult stemcells into the circulation.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.Cells transduced by the vector as described above in the context of exvivo therapy can also be administered parenterally as described above,except that lyophilization is not generally appropriate, since cells aredestroyed by lyophilization. The dose administered to a patient, in thecontext of the present invention should be sufficient to effect abeneficial therapeutic response in the patient over time. The dose willbe determined by the efficacy of the particular cells employed and thecondition of the patient, as well as the body weight of the patient tobe treated. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side effects that accompanythe administration of a cell type in a particular patient. Indetermining the effective amount of cells to be administered in thetreatment or prophylaxis of diseases, the physician should evaluatecirculating plasma levels, and, in the case of replacement therapy, theproduction of the gene product of interest.

Transduced cells are prepared for reinfusion according to establishedmethods. See, Abrahamsen et al., J. Clin. Apheresis 6:48-53 (1991);Carter et al., J. Clin. Apheresis 4:113-117 (1988); Aebersold et al., J.Immunol. Methods 112: 1-7 (1988); Muul et al., J. Immunol. Methods101:171-181 (1987) and Carter et al., Transfusion 27:362-365 (1987).After a period of about 2-4 weeks in culture, the cells may numberbetween 1×10⁶ and 1×10¹⁰. In this regard, the growth characteristics ofcells vary from patient to patient and from cell type to cell type.About 72 hours prior to reinfusion of the transduced cells, an aliquotis taken for analysis of phenotype, and percentage of cells expressingthe therapeutic agent.

For administration, cells of the present invention can be administeredat a rate determined by the LD₅₀ of the cell type, and the side effectsof the cell type at various concentrations, as applied to the mass andoverall health of the patient. Administration can be accomplished viasingle or divided doses. Adult stem cells may also be mobilized usingexogenously administered factors that stimulate their production andegress from tissues or spaces, that may include, but are not restrictedto, bone marrow or adipose tissues.

The invention has been described in detail with reference to preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements within the spirit and scope of theinvention. The following non-limiting examples are illustrative of theinvention.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, Applicants donot admit any particular reference is “prior art” to their invention.

EXAMPLES

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

Materials and Methods

Mesenchymal Stem Cell Isolation, Harvest and Labeling

Swine MSCs were isolated and expanded from a single, healthy maleYorkshire donor (Schuleri, K. H. et al. Am. J. Physiol Heart Circ.Physiol 294, H2002-H2011 (2008)). Briefly, bone marrow was obtained fromthe iliac crest, and aspirates were passed through a density gradient toeliminate undesired cell types and were plated with 25 ml MEM Alphamedia (Mediatech, Manassas, Va.) containing 20% fetal Bovine Serum(Hyclone, Logan, Utah.) in 162 cm² culture flasks (Fisher Scientific,Pittsburgh, Pa.). At 5-7 days after plating, non-adherent cells werewashed away during medium changes and the remaining, plastic adherent,purified MSC population was expanded in culture. The MSC population wasthen harvested and transduced with 5-Bromodeoxyuridin (BrdU) or greenfluorescent protein (Lenti-GFP vector, Lentigen) according tomanufacturers' instructions. All used cells were harvested when theyreached 80-90% confluence at passage 5. Labeled MSCs were placed in acryopreservation solution consisting of 10% DMSO, 5% porcine serumalbumin, and 85% Plasmalyte. Cells were placed in cryo bags at aconcentration of 5-10 million MSCs per ml and frozen in a control-ratefreezer to −180° C. until the day of implantation. By using trypan bluestaining, the viability of all thawed MSC lots was verified to be >85%before use in the study.

Induction of Myocardial Infarction and Transendocardial Injections

Seventeen healthy female Yorkshire swine weighed 25-35 kg, and 21healthy Gottingen miniature swine weighing 20-27 kg were included inthis study. Experimental myocardial infarction was generated (Schuleri,K. H. et al. Am. J. Physiol Heart Circ, Physiol 294, H2002-H2011 (2008);Amado, L. C. el al. J. Am. Coll. Cardiol. 48, 2116-2124 (2006)).Briefly, the right common carotid artery was canulated under anesthesiainduced with ketamine (33 mg/kg, IM) and maintained with isoflurane(1.5-2.0%). Myocardial infarction was induced by accessing the LeftAnterior Descending (LAD) coronary artery and occluding it after thefirst diagonal branch by inflating a coronary angioplasty balloon(2.75×15 mm). Because of differences in the coronary anatomy betweenGottingen and Yorkshires, the balloon was inflated for 150 min and 60min respectively in order to achieve comparable infarct sizes.Subsequently, the balloon was deflated, reperfusion was established andthe carotid artery was permanently closed. All animals were adequatelyheparinized during the procedure. The Yorkshire group of animalsreceived intramyocardial injections of allogeneic GFP labeled porcineMSCs (75×10⁶ cells) or Placebo (Plasmalyte alone, Baxter EdwardsCritical Care, Deerfield, Ill.), three days after myocardial infarction(MI). For the chronic heart failure model, infarcted minipigs weremonitored for 12 weeks before transplantation (total of 200×10⁶allogeneic BrdU-labeled MSCs or plasmalyte). All injections wereperformed under fluoroscopy, with a pistol-needle tip injection catheteradvanced to the LV through a steerable guide catheter (Stiletto, BostonScientific, Natick, Mass.). Hypokinetic, akinetic, and dyskinetic areaswere identified during contrast ventriculography, and injections wereperformed within and at the borders of the dysfunctional area, asdefined by bi-plane ventriculography. A total of 15 injections wereperformed in each animal, with each injection containing 0.5 ml of theinjectate. Each injection was fluoroscopically guided to distributecells evenly throughout the entire infarct and border zones.

Cardiac MRI

Cardiac MR imaging (CMR) of the Gottingen's heart function wereperformed at the following time points: baseline, 10 days, 1, 2 and 3months post-MI and 1, 2 and 3 months following transplantation. SerialCMR images were acquired with a four channel phase array, 1.5 T MRScanner (Siemens Symphony, Erlangen, Germany) in anesthetized animalswith electrocardiography gating and short breath-hold acquisition. Theprotocol for cine-CMR images and tagging-CMR images has been describedbefore (Amado, L. C. et al. J. Am. Coll. Cardiol. 48, 2116-2124 (2006);Amado, L. C. et al. Proc. Natl. Acad Sci. U.S.A. 102, 11474-11479(2005)). Briefly, LV Global function was assessed in steady state freeprecession with a number of slices to cover the entire LV from apex tobase. Imaging parameters were as follow: Echo delay time (TE)=1.9 ms,repetition time (TR)=4.2 ms; flip angle 45°; 256×160 matrix; 8 mm slicethickness/no gap; 28 cm field of view (FOV) and 1 number of signalaverage (NSA). Cine images were analyzed with research comprehensivesoftware validated by the Cardiology MR group at Lund University,Sweden.

To assess regional cardiac function, tagging MRI images were acquiredwith an ECG-gated, segmented k-space, fast gradient recall echo-pulsedsequence with a spatial modulation to generate a grid pattern. Imageswere obtained at the same level of cine-MR images with the followingparameters: TR: 6.7, TE: 3., flip angle=12°; 256×160 matrix;views/second 4; 8 mm slice thickness no gap; 31.25 kHz; 28 cm FOV; 1NSA; and 6 pixels tagging space. Images were quantitatively analyzedwith a custom software package (DIAGNOSOFT™ PLUS, Diagnosoft Inc, PaloAlto, Calif.). Regional strain magnitude was determined from the 24radially displaced segments for each short axis section covering theentire LV and averaged among slices for each region and each time point,generating a strain map for each time point over the cardiac cycle. Thepeak systolic circumferential strain (peak Ecc) was determined from thestrain map for each time point Delayed Contrast enhancement was used toassess the extension of the infarcted area. The protocol included anintravenous bolus of Gadolinium-DTPA (0.1 mmol/kg, 5 m/s, MAGNEVIST™,Berlex, Bayer Healthcare Pharmaceuticals) through a peripheralintravenous line. Images were acquired 15 minutes later at the samelocation as the short axis cine-images. Imaging parameters were TR=7.3,TE=3.3, TI=200 ms; flip angle=25°, 256×196 matrix; 8 mm slice thicknessgap 31.2 kHz, 28 cm FOV and 2 NSA.

Histology

For the first phase of the study, microscopic evaluation between thetreated (n=3), placebo (n=3) and control (n=3) Yorkshire pigs wasperformed at 2 weeks after the intramyocardial injections. Moreover, inorder to assess the acute immunophenotypic evolution of the transplantedMSCs, 8 more animals were sacrificed at 24 h (n=1 placebo and n=1 MSCstreated) and 72 h (n=3 placebo and n=3 MSCs treated) postinjections. Forthe second phase of the study, microscopic evaluation between thetreated (n=12) and placebo (n=9) Gottingen pigs was performed 3 monthsafter the intramyocardial injections. All animals completing the studywere humanely euthanized and their hearts were excised, sectioned into4-mm-thick short-axis slices, weighted and digitally photographed.Multiple tissue samples were collected from the infarcted, border andremote zones of each slice, fixed in 10% buffered formalin and embeddedin paraffin. Hematoxylin and Eosin (H&E), as well as Masson's Trichromestaining were used for the primary histological examination.

Immunofluorescence Confocal Microscopy

Immunofluorescence studies were carried out on 4 μm-thick paraffinsections. Briefly, after deparaffinizing and rehydrating the tissuesections, antigen unmasking was performed by microwaving the slides for20 min in citrate buffer Solution, pH=6 (Dako, Carpenteria, Calif.). Thesections were blocked for 1 h at RT with 10% normal donkey serum(Chemicon International Inc, Temecula, Calif.), followed by 1 hincubation at 37° C. with the primary antibody. The following antibodieswere used: C-kit, α-sarcomeric actinin, α-smooth muscle actin,Connexin-43 (Sigma, Saint Louis, Mo.), N-cadherin, anti-GFP, Laminin,Phospho-Histone H3 (Abeam, Cambridge, Mass.), GATA-4, MDR1, Integrin-β1,CD3, CD14, CD68 (Santa Cruz Biotechnologies, Santa Cruz, Calif.),activated Caspase-3 (ED Biosciences, San Jose, Calif.), Nkx2.5 (R&Dsystems Inc, Minneapolis, Minn.), Factor VIII (Biocare Medical, Concord,Calif.) and KDR (Cell Signaling, Boston, Mass.). Consequently, theantibodies were visualized by incubating the sections for 1 h at 37° C.with FITC, Cy3 and Cy5-conjugated F(ab′)₂ fragments of affinity-purifiedsecondary antibodies (Jackson Immunoresearch, West Grove, Pa.). Slideswere counterstained with DAPI, mounted with ProLong Antifade Goldreagent (Invitrogen, Carlsbad, Calif.) and stored at 4° C. until furtherexamination. Microscopic evaluations and image acquisitions wereperformed with a Zeiss LSM-510 Confocal Microscope (Carl ZeissMicroImaging, Inc. Thomwood, N.Y.).

Tissue Mapping

For assessing the trafficking of the MSCs, the Compucyte Laser ScanningCytometer (LSC) was used according to manufacturers' instructions(CompuCyte Corporation, Cambridge, Mass.). Briefly, after detecting andamplifying the signal of the GFP^(pos) transplanted MSCs with aFITC-labeled anti-GFP antibody according to immunofluorescent protocolsestablished in this laboratory, the tissue slides were scanned under theLSC to map the spatial distribution of the FITC signal on the samples.The detected fluorescent signal was projected as a scatter plot andaccordingly, was adjusted and merged to the H&E scanned slide (Coolscan,Nikon) of the same sample by using an imaging software package (AdobePhotoshop CS3). The detected scatters were further cross-checked underthe confocal microscope to exclude the detection of any false positivesignal.

Fluorescence in Situ Hybridization

Fluorescence in Situ Hybridization (FISH) was employed to detect the Ychromosome of the sex-mismatched transplanted allogeneic MSCs in thefemale porcine hearts. The metaphase chromosomes were detected byhybridizing the tissue samples with Cy3-conjugated porcine Y chromosomepaints (StarFISH, Cambio Ltd, Cambridge, UK) according to manufacturers'instructions. Briefly, following deparaffinization and rehydration, thesamples were microwaved for 20 min in citrate Buffer, pH=6 (Dako). Aftercooling for 30 min at room temperature (RT), tissues were digested for 3min at 37° C. with pepsin, washed with 2×SSC buffer (Invitrogen) anddehydrated through serial ethanol washing steps. The samples wereair-dried and the probe was applied. After covering the samples with acover slip and sealing them with rubber cement, the samples were placedin the hybridizer (Dako) for denaturation (10 min at 80° C.) followed byovernight hybridization at 37° C. The next day, samples were washed with2×SSC, mounted with DAPI and covered.

Morphometric Analyses and Microscopic Evaluation

The numbers of GFP^(pos), c-kit^(pos) and Y^(pos) cells were quantitatedper square millimeter (mm²). Apoptotic and mitotic cells at 24 h and 72h time points were expressed as the percentage of the GFP^(pos) cellsco-localized with activated caspase 3 and phospho-H3 respectively. Thesame approach was followed for the quantification of the cardiac andvascular commitment of the allografts as well as the endogenousc-kit^(pos) cells. Morphometric analysis was performed by using a customresearch package (Image J, NIH, Bethesda, Md.). For assessing thedensity of newly formed vessels, the axial ratio (major diameter/minordiameter) for each Y chromosome containing vascular structure wasobtained. This methodology was used to correct the angle of orientationof the vessels with the plane of section (Tillmanns, J. et al. Proc.Natl. Acad. Sci. USA 105, 1668-1673 (2008)). The sum of the axial ratiosof vessels per unit area of tissue yielded the vessel density persample.

Statistical Analysis

All the values are presented as means±SEM. All analyses were performedby using the SPSS for Windows version 15.0 (SPSS Inc., Chicago, Ill.).Differences between groups following immunohistological evaluation werecompared by using One Way ANOVA. Differences between groups in ejectionfraction and infarct size based on MR images were calculated by usingone-way ANOVA within different time points. The Tukey's test was usedfor the past-hoc analysis. Difference in peak Ecc were calculated byrepeated measurements 2 way analysis of variance with one factorrepetition. Pearson correlation was applied to find a relation betweencell engraftment and increase in peak Ecc. A level of P≤0.05 wasconsidered statistically significant.

Animal Studies

For this study, 17 healthy female Yorkshire swine weighing 25-35 kg, and21 healthy Gottingen miniature swine weighing 20-27 kg, underwentexperimental myocardial infarction (MI) followed by reperfusion. Thestudy was conducted in 2 phases. In the first phase, three groups werestudied: Yorkshire pigs received transendocardial injections (TED(Stiletto, Boston Scientific, Natick, Mass.) of 75×10⁶ GFP labeledallogeneic adult bone marrow derived mesenchymal stem cells (αMSCs)(n=7), Placebo (n=7) or no injection (n=3) three days following the MI.Animals were sacrificed at 24 h (n=1 placebo and n=1 αMSCs treated), 72h (n=3 placebo and n=3 αMSCs treated) and 2 weeks (n=3 αMSCs treated n=3placebo and n=3 control) after transplantation in order to study thefate of the allogeneic cells.

In the second phase, two groups were studied: Gottingen minipigsreceived TEI of 200×10⁶ cells of male αMSCs (n=12) or placebo (n=9), 12weeks after MI. Animals were followed up by cine and Tagging-MRIanalyses (Siemens Symphony, Erlangen, Germany) at multiple time points(baseline, 10 days, 1, 2 and 3 months post-MI and 1, 2 and 3 monthsfollowing transplantation) in order to assess the amount of functionalrecovery of the treated vs. the untreated groups.

Example 1 Mesenchymal Stem Cells Regenerate Cardiac Muscle, Vasculatureand Stem Cell Niches

In order to determine whether mesenchymal stem cells have cardiacprecursor potential or whether their therapeutic effect is exertedthrough paracrine effects, GFP labeled male allogeneic adult bone marrowderived mesenchymal stem cells (αMSCs) were injected into female pigsfollowing myocardial infarction (MI) (FIG. 5). Without wishing to bebound by theory, it was hypothesized that allogeneic adult bone marrowderived mesenchymal stem cells (αMSCs) are true adult precursor cellswhich can differentiate into the three cardiac cell lineages, and thatαMSCs participate in cardiac recovery by forming cell-cell interactionswith existing myocardial elements including with endogenous precursorcells.

First the fate of allogeneic GFP, Y-chromosome positive αMSCs injecteddirectly into the myocardium via an endocardial catheter in pigs 3-daysfollowing myocardial infarction (MI), was examined. After cell deliveryto the infarct and border zones, cell retention was robust and wasentirely confined to the infarct and border zones of the myocardium,with evidence of migration of cells throughout this region (FIGS. 6A-6Eand FIGS. 7A-7D), including endo- to epicardial migration, and incellular streams throughout the area of damage. From 1 to 3 days afterinjection, αMSCs displayed evidence of self-renewal with minimalapoptosis (FIGS. 8A, 8C). Immunostaining for phosphorylated histone-H3 1and 3 days after transplantation demonstrated that a substantial numberof cells entered the cell cycle and underwent mitosis, whereasconcurrent absence of the pro-apoptotic marker activated caspase-3 inthe majority of αMSCs indicated their capacity for survival (FIGS. 8B,8C).

To address lineage commitment of αMSCs we assessed co-localization ofGFPpos αMSCs with the cardiac transcription factor GATA-4, endothelialmarkers Factor VIII and KDR, the vascular smooth muscle marker α-smoothmuscle actin, and the cardiomyocyte marker α-sarcomeric actinin. MSCsdifferentiation into cardiac precursor cells was evident 24 h aftertransplantation and increased 2-fold by 72 hours, an increase driven byboth differentiation as well as cell replication (FIGS. 1A, 1B, FIG.8C). In addition, endothelial and vascular smooth muscle cell lineagecommitment was not present 24 h after transplantation, but could both bedetected by 72 hours (FIGS. 1C-1F). There were GFP^(pos) αMSCs(1.3±0.4%) that co-localized with KDR/Factor VIII, documenting lineagecommitment to endothelial cells. There was extensive cell-cellinteraction between GFP^(pos) αMSCs and native cardiac cells, mediatedby Connexin-43 and N-cadherin FIGS. 1G, 1H).

Two weeks after injection, there remained robust cell retention andfurther differentiation into mature cell phenotypes (FIG. 2, FIGS.7A-7D]. GFP^(pos) αMSCs expressed α-sarcomeric actinin and GATA-4 andexhibited morphological characteristics both of immature cardiacprecursor cells FIGS. 2A, 2B) and of mature cardiac myocytes andvascular cells (FIGS. 2C, 2D). Chimeric myocardium was present withininfarct and border zones but not in the remote areas, and formedcell-cell interactions with native cardiac myocytes (FIGS. 2E, 2F).

Next, it was tested whether αMSCs interact with endogenous c-kit^(pos)cardiac stem cells (CSCs). Endogenous c-kit^(pos) CSCs were rare 1-3days after transplantation, but dramatically increased in the αMSCstreated pigs by 2 weeks (FIGS. 2G, 2H). The number of c-kit cellsincreased by 50-fold in the infarct zone of the treated animals and wasalso elevated, though less dramatically, in border and remote zones ofMSC treated animals (FIGS. 9A-9D). These c-kit^(pos) cells wereCD3^(neg), CD14^(neg) and CD68^(neg), excluding inflammatory and mastcells phenotypes, and were detected in clusters in the infarcted andborder zones of the αMSCs treated animals. In remote areas and innon-treated hearts, c-kit cells were observed as a rare isolated celland were not part of cell clusters (FIGS. 2G, 2H, and FIG. 7C and FIGS.9A-9D]. To address whether endogenous c-kit cells were coupled to hostor chimeric cells, the expression of connexin-43 and N-cadherin wastested; c-kit cells formed gap junctions and mechanical connections viathese proteins with other c-kit cells as well as with GFP^(pos) MSCs(FIGS. 2I, 2J). These cell-cell interactions closely resemble cardiacstem cell niches. Further immunohistochemical characterizationdemonstrated that a number of these cells co-expressed MDR1 and GATA-4,indicating the commitment to cardiac lineage within the regeneratedniches. There was a 25-fold increase in the number of these cardiaclineage committed c-kit cells in the border zones of the αMSCs treatedanimals, compared to animals receiving injection of non-cellular vehiclealone, and to untreated control animals. No differences were noted inthe infarcted and non-infarcted zones, indicating the presence of anactive endogenous repair mechanism in the border zones of the treatedhearts (FIGS. 10A-10C). These findings introduce a novel mechanismunderlying MSC treatment which, in addition to regenerating chimericmyocardial tissue within the first two weeks after transplantation, alsonow is shown to orchestrate a program of endogenous cardiac repaircomprising an influx of c-kit^(pos) cells with lineage commitment to themyocytes phenotype and through the formation of structures resemblingcardiac stem cell niches.

Next long-term αMSC engraftment and contribution to functional cardiacrecovery, late after infarction was examined. BrdU-labeled male αMSCswere injected into female animals that had sustained myocardialinfarction 3 months prior. These animals were studied for 3 monthsfollowing cell injection by serial cardiac MRI, followed by histologicevaluation. Three months after MI, pigs had diminished ejectionfractions (33.4±1.0% vs 47.7±2.0% at baseline; p=0.008), the infarctionscar comprised 18.3±2.5% of the ventricle, and significant regionaldysfunction of the scar segment was documented using MR tagging (FIGS.3A-3J). Intramyocardial injection of αMSCs three months after MIproduced substantial increases in ejection fraction, reductions in scar,and restored regional cardiac function in the border zones of the scarcorresponding to the regions of scar reduction (FIGS. 3A-3H).Importantly, the reduction in scar size correlated closely with regionalfunctional recovery (FIG. 3H).

The hearts were analyzed by immunohistologic examination to test whetherthis functional recovery required cell engraftment. Y chromosomecontaining cells (Y^(pos)) were present in the infarct and border zonesof the αMSC-treated group, but were not detected in the placebo group,nor in the remote zones of the αMSC-treated animals. The density ofY^(pos) cells did not differ between the infarct zone (0.17±0.04cells/mm²) and border zone (0.15±0.03 cells/mm²) (FIGS. 11A, 11B)Co-staining with cardiac-specific transcription factor GATA-4 andα-sarcomeric actinin showed that 14.0±4.0% of Y^(pos) cells had an adultcardiac myocyte phenotype. Interestingly, Y^(pos) cells of donor originalso exhibited putative gap junction formation with residentcardiomyocytes, as indicated by expression of connexin-43 (FIG. 11C).Importantly, the total number of Y^(pos) cells engrafted in the BZcorrelated with the improvement in the peak systolic circumferentialstrain (Ecc) (r=0.8; p=0.03) (FIG. 3I), indicating a criticalcontribution of cell engraftment and differentiation to the degree offunctional recovery.

Y^(pos) cells were also present in both large and small vascularstructures, supporting participation in new vessel formation in IZ andBZ. Donor cells differentiated into both vascular muscle and endotheliallineages, as evidenced by co-localization of α-smooth muscle actin andfactor VIII, respectively (FIGS. 4A, 4C). An overall of 9.9±2.4% of theYpos cells were incorporated into vessel walls, with 5.9±1.9%co-expressing Factor VIII and 4.0±1.6% α-smooth muscle actin. Thedensity of newly-formed vessels was calculated in vessel length percubic millimeter. In IZ, the density of the cells was 0.05±0.01 mm/mm³and in the BZ it was similarly 0.04±0.02 mm/mm³ (FIG. 4D). In BZ αMSCswere incorporated mostly in large and medium size vessels (500 μm-1 mm,FIGS. 4A, 4B, 4D, 4E) composed of both endothelial and smooth musclelayers, illustrating their capacity to restore blood flow through largecoronary vessels formation. In IZ, most of the new vessels had <20 μmdiameter and were composed of single endothelial layers without smoothmuscle layers, consistent with capillary formation.

Not all patients with heart disease may be able to receive effectiveautologous cell-based therapies, since age and host diseases may affectthe quality of the autologous grafts per se. MSCs on the other hand, area true stem cell population with known immunomodulatory andimmuno-tolerogenic capacities that are a safe and successful cardiacallograft. MSCs meet alI the expectations of a cell product that can bereadily available in therapeutic quantities, thus providing an “off-theshelf” cell-based therapy for treating heart disease.

Together these findings demonstrate that allogeneic MSCs, unaltered toenhance their survival, engraft and exhibit trilineage cardiac celldifferentiation. The repair process is driven by damage signals, andoccurs both in acute and in chronic myocardial infarction. MSCengraftment is accompanied by the formation of cell-cell interactionsboth with host adult cells and with host endogenous precursor cells. Thelatter reconstitutes multi-cellular clusters that have the features ofstem cell niches and promotes the migration and myocytic differentiationof endogenous cardiac precursor cells. Within 72 h the fate of theallografts is determined, and the level of regeneration is preserved upto three months after transplantation. The degree of engraftmentcorrelates with recovery of cardiac function, which is substantial, andwith reduction in myocardial scar formation. While scar reduction isincomplete, these findings raise the possibility that strategiesinvolving repeated αMSCs applications or acceleration of theirdifferentiation rates could result in near total elimination of cardiacscar. The public health impact of an effective treatment for chronicischemic cardiomyopathy is immense, and these data are the first toindicate such a potential for MSC therapy. The totality of thesefindings document an impressive capacity of an adult bone marrow derivedstem cell to repair acutely and chronically injured hearts, and haveimportant mechanistic and clinical implications.

Example 2 Bone Marrow Mesenchymal Stem Cells Stimulate Cardiac Stem CellProliferation and Differentiation

Methods

This study was reviewed and approved by the University of MiamiInstitutional Animal Care and Use Committee and complies with allFederal and State guidelines concerning the use of animals in researchand teaching as defined by The Guide For the Care and Use of LaboratoryAnimals (NIH Pub. No. 80-23, revised 1985).

Mesenchymal Stem Cell Isolation, Harvest and Labeling:

Swine MSCs were isolated and expanded from a single, healthy maleYorkshire donor as previously described. Briefly, bone marrow wasobtained from the iliac crest, and aspirates were passed through adensity gradient to eliminate undesired cell types and were plated with25 ml MEM Alpha media (Mediatech, Manassas, Va.) containing 20% fetalBovine Serum (Hyclone, Logan, Utah) in 162 cm² culture flasks (FisherScientific, Pittsburgh, Pa.). At 5-7 days after plating, non-adherentcells were washed away during medium changes and the remaining, plasticadherent, purified MSC population was expanded in culture. The MSCpopulation was then harvested and transduced with 5-Bromodeoxyuridin(BrdU) or green fluorescent protein (Lenti-GFP vector, Lentigen)according to manufacturers' instructions. All used cells were harvestedwhen they reached 80-90% confluence at passage. Labeled MSCs were placedin a cryopreservation solution consisting of 10% DMSO, 5% porcine serumalbumin, and 85% Plasmalyte. Cells were placed in cryo bags at aconcentration of 5-10 million MSCs/ml and frozen in a control-ratefreezer to −180° C. until the day of implantation. By using trypan bluestaining, the viability of all thawed MSC lots was verified to be >85%before use in the study.

Organotypic Cultures:

Cardiac biopsies were collected from the right ventricular septal wallof 7 Yorkshire swine with or without myocardial infarction. The biopsieswere harvested and kept in cold Hank's Balanced Salt Solution (Lonza)containing 1% penicillin/streptomycin until processing. After washingthoroughly with DMEM (GIBCO), samples were minced in ˜1 mm³ cubes anddigested in a solution of DMEM/F12 (GIBCO), 20% FBS, 1%penicillin/streptomycin and 200 units/ml Collagenase-Type II solution(Worthington) at 37° C. for 3 h. Following that, whole lysates werecollected, washed twice with DMEM, resuspended in DMEM/F12, 20% FBS, 1%penicillin/streptomycin and plated in T-25 tissue culture flasks(Corning) that contained 2-b 3×10 ⁵ GFP+ porcine MSCs or not. After 1week, samples were collected by trypsinization and c-kit⁺ cells werepurified by repeated immune panning in a Petri dish as previouslydescribed. After 2-3 days in culture, the isolated cells weretrypsinized and re-plated in a Petri dish containing F12K (GIBCO), 5%PBS, 10 ng/ml bFGF (peprotech), 20 ng/ml LIF (Sigma) and 1%penicillin/streptomycin where they grew for 7-10 days. Next, only thenon-adherent fraction was collected and expanded as semi-adherent cellsin tissue culture dishes containing DMEM/F12, 2% FBS,Insulin-Transferrin-Selenite (Sigma), 10 ng/ml bFGF, 10 ng/ml LIF, 20ng/ml EGF (peprotech), 100 ng/ml SCF (Peprotech) and 1%penicillin/streptomycin. Subsequent immunocytochemical evaluation wasperformed on cytospin preparations according to manufacturers'instructions (Sakura Finetek).

In Vitro Differentiation Assays:

To test the differentiation capacity of CPCs into cardiac myocytes,co-cultures were performed with neonatal rat cardiac myocytes (NRCMs).Briefly, NRCMs were isolated as previously described and plated at adensity of 1×10⁵ NRCMs/cm² in 12-well plates (Corning) containingcollagen-coated glass coverslips. CPCs were then co-cultured with NRCMsin a ⅓ ratio, directly or indirectly using transwell inserts with a 0.4μm pore size (BD)). Cocultures were maintained with NRCM mediumconsisting of DMEM (GIBCO), insulintransferrin-selenite (Sigma), 2 mg/mlbovine serum albumin, 20 μg/ml ascorbic acid, 1% penicillin-streptomycinand incubated for up to 2 weeks in humidified incubator at 37° C. and 5%CO₂. For immunocytochemical evaluation, cells were fixed in 4%paraformaldehyde for 20 min at RT, 24 h, 72 h and 2 weeks after plating.

Induction of Myocardial Infarction and Transendocardial Injections:

Thirty one healthy female Yorkshire swine weighed 25-35 kg, wereincluded in this study. Experimental myocardial infarction was generatedaccording to our previously described protocols. Briefly, the rightcommon carotid artery was canulated under anesthesia induced withketamine (33 mg/kg, IM) and maintained with isoflurane (1.5-2.0%). MIwas induced by accessing the Left Anterior Descending (LAD) coronaryartery and occluding it after the first diagonal branch by inflating acoronary angioplasty balloon (2.75×15 mm) for 60 min followed byreperfusion. All animals were adequately heparinized during theprocedure. The study was conducted in 2-phases. In the first phase, itwas sought to explore the mechanisms underlying MSCs-based cardiacrepair, therefore animals received intramyocardial injections ofallogeneic GFP labeled porcine MSCs (75×10⁶ cells) or Placebo(Plasmalyte alone, Baxter Edwards Critical Care, Deerfield, Ill.), threedays after MI.

The second phase was designed to address whether MSCs-implantation isnecessary for successful cardiac repair or their secreted factors alonecould exert similar effects; therefore animals were randomized toreceive intramyocardial injections of allogeneic GFP labeled porcineMSCs (100×10⁶ cells) or the rich in secreted factors conditioned mediumwere the MSCs had been expanded into, concentrated 10× (Stirred Cell,Millipore). All investigators involved in this study were blinded. Allinjections were performed under fluoroscopy, with a pistol-needle tipinjection catheter advanced to the LV through a steerable guide catheter(Stiletto, Boston Scientific, Natick, Mass.). Hypokinetic, akinetic, anddyskinetic areas were identified during contrast ventriculography, andinjections were performed within and at the borders of the dysfunctionalarea, as defined by bi-plane ventriculography. A total of 15 injectionswere performed in each animal, with each injection containing 0.5 ml ofthe injectate. Each injection was fluoroscopically guided to distributecells evenly throughout the entire infarct and border zones.

Cardiac MRI:

For the second phase, therapeutic effect on cardiac function wasassessed by Cardiac MR imaging (cMRI). Cardiac structure and functionwere monitored at baseline, 1 day prior to injections, 4 days, 2 weeksand 8 weeks post-injections. Serial cMRI images were acquired with afour channel phase array, 1.5T MR Scanner (Siemens Symphony, Erlangen,Germany) in anesthetized animals with electrocardiography gating andshort breath-hold acquisition. The protocol for cine-cMRI andtagging-cMRI has been described before. Briefly, LV Global function wasassessed in steady state free precession with a number of slices tocover the entire LV from apex to base. Imaging parameters were asfollow: Echo delay time (TE)=1.9 ms, repetition time (TR)=4.2 ms; flipangle 45°; 256×160 matrix; 8 mm slice thickness/no gap; 28 cm field ofview (FOV) and 1 number of signal average (NSA). Cine images wereanalyzed with research comprehensive software validated by theCardiology MR group at Lund University, Sweden (segment.heiberg.se).

The protocol included an intravenous bolus of Gadolinium-DTPA (0.1mmol/kg, 5 m/s; MAGNEVIST™, Berle; Wayne) through a peripheralintravenous line. Images were acquired 15 minutes later at the samelocation as the short axis cine-images. Imaging parameters were TR=7.3,TE=3.3, TI=200 ms; flip angle=25°, 256×196 matrix; 8 mm slice thicknessgap 31.2 kHz, 28 cm FOV and 2 NSA.

Histology:

For the first phase of the study, microscopic evaluation between thetreated (n=3), placebo (n=3) and control (n=3) Yorkshire pigs wasperformed at 2 weeks after the intramyocardial injections. Moreover, inorder to assess the time course of MSCs engraftment and differentiation,8 more animals were sacrificed at 24 h (n=2 placebo and n=2 MSCstreated) and 72 h (n=3 placebo and n=3 MSCs treated) post-injections.For the second phase of the study, microscopic evaluation between theMSCs and Cx-treated pigs was performed at 2 weeks (n=3 each) and 8 weeks(n=3 each) after TEIs. All animals were humanely euthanized throughintravenous infusion with KCL to arrest the hearts in diastole. Theexplanted hearts were then washed in ice-cold phosphate buffer saline(PBS) to remove any residual blood, followed by perfusions through theleft and right coronary arteries with 10% buffered formalin. Heartchambers were then filled with dental impression material (Imprint, 3MESPE) to preserve heart's shape during fixation. The hearts were thenfixed for 24 h in 10% buffered formalin and sliced transversely intoseven to eight −4 mm thick slices using a commercial meat cutter,weighted and digitally photographed. Representative samples wereselected from the infarcted (IZ), border (BZ) and remote areas (RZ) ofeach slice, and embedded in paraffin (FFPE) for immunohistochemicalevaluation. Hematoxylin and Eosin (H&E), as well as Masson's Trichromestaining were used for the primary histological examination. Forconfocal immunofluorescence quantification, 4-5 μm thick FFPE slidesfrom each region (IZ, BZ, RZ) were evaluated. The total numbers ofpositively-stained cells were quantified per slide to calculate thenumber of cells per unit volume (cm³) on each sample. Morphometricanalysis was performed by using a custom research package (Image J, NIH,Bethesda, Md.).

Immunofluorescence Confocal Microscopy:

Immunofluorescence studies were carried out on 4 μm-thick paraffinsections, according to previously described protocols. Briefly, afterdeparaffinizing and rehydrating the tissue sections, antigen unmaskingwas performed by microwaving the slides for 20 min in citrate bufferSolution, pH=6 (Dalco, Carpenteria, Calif.). The sections were blockedfor 1 h at RT with 10% normal donkey serum (Chemicon International Inc,Temecula, Calif.), followed by 1 h incubation at 37° C. with the primaryantibody. The following antibodies were used: C-kit, α-sarcomericactinin, α-smooth muscle actin, α-smooth muscle myosin heavy chain,Connexin-43 (Sigma, Saint Louis, Mo.), N-cadherin, anti-GFP, Laminin,Phospho-Histone H3, cardiac troponin-I (Abeam, Cambridge, Mass.),GATA-4, MDR1, VE-cadherin, CD3, CD14, CD68 (Santa Cruz Biotechnologies,Santa Cruz, Calif.), activated Caspase-3 (BD Biosciences, San Jose,Calif.), Nkx2.5 (R&D systems Inc, Minneapolis, Minn.), FactorVIII-related antigen (Biocare Medical, Concord, Calif.), Isl-1 (40.2D6,Developmental Studies Hybridoma Bank, Iowa), cardiac myosin lightchain-2 (Novus Biologicals, Littleton, Co.) and KDR (Cell Signaling,Boston, Mass.). Consequently, the antibodies were visualized byincubating the sections for 1 h at 37° C. with FITC, Cy3 andCy5-conjugated F(ab′)₂ fragments of affinity-purified secondaryantibodies (Jackson Immunoresearch, West Grove, Pa.). Slides werecounterstained with DAPI, mounted with ProLong Antifade Gold reagent(Invitrogen, Carlsbad, Calif.) and stored at 4° C. until furtherexamination. Microscopic evaluations and image acquisitions wereperformed with a Zeiss LSM-510 Confocal Microscope (Carl ZeissMicroImaging, Inc. Thomwood, N.Y.). The Zeiss Axiovision software(release 4.7.1.0, Carl Zeiss Imaging Solutions, GmbH) was used for 3Drendering of the confocal Z-stack images.

Fluorescence in Situ Hybridization:

Fluorescence in Situ Hybridization (FISH) was employed to detect theY-chromosome of the sex-mismatched transplanted allogeneic MSCs in thefemale porcine hearts. The Y-chromosome containing cells were detectedby hybridizing the tissue samples with Cy3-conjugated porcine Ychromosome paints (StarFISH, Cambio Ltd, Cambridge, UK) according tomanufacturers' instructions. Briefly, following deparaffinization andrehydration, the samples were microwaved for 20 min in citrate Buffer,pH=6 (Dako). After cooling for 30 min at RT, tissues were digested for 3min at 37° C. with pepsin, washed with 2× SSC buffer (Invitrogen) anddehydrated through serial ethanol washing steps. The samples wereair-dried and the probe was applied. After covering the samples with acoverslip and sealing them with rubber cement, the samples were placedin the hybridizer (Dako) for denaturation (10 min at 80° C.) followed byovernight hybridization at 37° C. The next day, samples were washed with2×SSC, mounted with DAPI and covered as previously described.

Statistical Analysis:

All the values are presented as means±SEM. All analyses were performedby using the SPSS for Windows version 15.0 (SPSS Inc., Chicago, Ill.).Differences between groups following immunohistological evaluation werecompared by using One Way ANOVA. Differences between groups in ejectionfraction and infarct size based on cMRI were calculated by using two-wayrepeated measures ANOVA. The Tukey's test was used for the post-hocanalysis. A level of P≤0.05 was considered statistically significant.

Results:

Although the heart has regenerative potential, it is insufficient torestore functioning myocardium after injury. Cell-based therapies intentto overcome this limited endogenous repair capacity, by directlyreplacing damaged tissue. Without wishing to be bound by theory, it washypothesized here, that they could also ameliorate myocardialsenescence. The data show that bone marrow mesenchymal stem cells (MSCs)engraft and differentiate into cardiomyocytes, but to a much greaterextent stimulate innate cardiopoietic mechanisms. MSCs couple with hostmyocardium, participate in cell-cell interactions, and enhanceendogenous c-kit⁺ cardiac precursor cell (CPCs) amplification andcardiac lineage commitment. In vitro, MSCs stimulate c-kit⁺ CPCs toproliferate into enriched populations of adult cardioblasts from whichdifferentiated spontaneously contracting cardiomyocytes arise. MSCs canbe used to stimulate host CPCs, a new mechanism of action underlyingsuccessful cell-based therapeutics.

It was also hypothesized herein, that MSCs stimulate cardiac repairthrough cell-autonomous effects that stimulate host myocardial precursorcells to amplify and differentiate into cardiomyocytes. To address thisprediction, 100×10⁶ GFP-labeled, male porcine MSCs were injected intothe infarct and border zone in female pigs 3 days following myocardialinfarction (MI); another group received injection of concentratedconditioned medium (CCM), so as to test whether secreted factors alonewould be sufficient to stimulate host cardiac repair.

As shown by serial cardiac magnetic resonance imaging (cMRI), MI wasaccompanied by a reduced ejection fraction (EF) [27.9±1.13% and25.8±3.1% for MSC and CCM groups respectively, p=NS, p<0.001 vsbaseline] and scar tissue that comprised ˜25% of the left ventricle(25.4±2.2% and 24.7±2.9% of the left ventricles of the MSC and CCMgroups, respectively, p=NS) (FIGS. 12A-12D). MSC-treatment decreasedinfarct size as early as 4 days after implantation achieving ˜50%reduction in scar size by 8 weeks. By 2 weeks EF had improvedsignificantly, reaching normal function by week 8 (FIGS. 12A-12D). Incontrast, treatment with CCM did not produce significant structural orfunctional improvements, indicating a dependence upon the cells forsustained cardiac repair.

Confocal immunofluorescence was employed to detect GFP and Y chromosomelabeled cells in infarct (IZ) and border (BZ) zones of the myocardium(FIGS. 16A-16D) and 1,585±746 and 1,317±393 cells/cm³ were detected 1and 3 days post-implantation, respectively. As early as 1 day afterinjection, MSCs entered the cell cycle, evidenced by the mitotic markerof serine-10 phosphorylated histone-H3 (Phospho-H3), and exhibitedminimal apoptosis, as evidenced by the pro-apoptotic marker activatedcaspase-3 (FIGS. 16A-16D).

While MSCs lacked of any marker of cardiovascular lineage in vitro,their commitment into cardiomyocytes began to occur within 24 hours andby 2 weeks they had differentiated into new, mature cardiomyocytes andvascular structures (FIGS. 13A-13E). Chimeric myocardium was detectedthroughout IZ and BZ but not in the remote zones of the treated hearts(FIGS. 13A-13E). Quantification of Y-chromosome containingcardiomyocytes illustrated that the lineage commitment of MSCs waspersistent from 3 days throughout the 2 month period of the study, andsimilar numbers of MSCs that had differentiated into myocytes weredetected during this time (FIGS. 13A-13E; FIG. 14A). Immunophenotypiccharacteristics of porcine MSCs before transplantation were shown byimmunocytochemical staining of the porcine MSCs illustrating theirnative phenotype; all cells were negative for markers such as GATA-4,KDR, isl-1, CD68, MDR1 and c-kit. Porcine Peripheral Blood MononuclearCells (PBMCs) used as a control cell type, for evaluating theaforementioned markers. PBMCs were negative for GATA-4 and Isl-1, butcontained positive fractions for KDR, c-kit, CD68 and MDR1.

Next, the impact of MSCs on endogenous CPCs was addressed, as identifiedby the expression of the stem cell factor-receptor, c-kit. It was firstconfirmed that the detected c-kit⁺ cells lacked GFP or the y-chromosome,confirming their endogenous origin (FIGS. 14A-14H). Quantification ofc-kit⁺ cells in infarct hearts illustrated sporadic distribution duringthe first 3 days after transplantation that was not different betweenMSC and CCM groups. However, 2 weeks after the transendocardialinjections, endogenous c-kit⁺ CPCs increased 11-fold in MSC but not CCMtreated animals (FIGS. 14A-14H; FIGS. 17A-17F). Importantly, CPCs weredetected in clusters in the IZ and BZ of MSC treated animals, but theywere found as rare isolated cells in remote areas as well as in thenon-MSC treated hearts (FIGS. 14A-14H; FIGS. 17A-17F). In addition, CPCsformed potential gap junctions and mechanical connections with otherc-kit⁺ cells, adult cardiomyocytes, and with GFP⁺ MSCs (FIGS. 14A-14H;FIGS. 17A-17F), and formed structures that resemble cardiac stem cellniches. Further immunohistochemical characterization demonstrated that anumber of CPCs coexpressed MDR1 and GATA-4, indicating a cardiac lineagecommitment (FIGS. 14A-14H). The number of cardiac committed CPCs was2-fold increased in the IZ and 15-fold increased in the BZ ofMSC-treated animals compared to animals receiving either CCM,non-cellular vehicle alone, and untreated controls. There were nodifferences between groups in the non-infarcted zones, indicating thepresence of an active endogenous repair mechanism in the damaged zonesof the treated hearts (FIGS. 14A-14H).

In vitro experiments were next performed to study the function ofendogenous CPCs (FIGS. 15A-15K ₁). The origin of c-kit⁺ CPCs as well astheir association to MSCs, was examined in a vivo organotypic co-cultureexperiments. Fresh or cryopreserved endomyocardial biopsies from porcinehearts were cultured for one week with or without MSCs. In additionalcontrol experiments, MSCs were cultured under the same conditionswithout a myocardial biopsy. After 3 days, myocardial biopsies becameinfiltrated by MSCs and adhered to the MSC monolayers. In contrast,biopsies cultured without MSCs remained in suspension (FIG. 15B, 15C).Within one week organotypic co-cultures became confluent, andpurification by repeated immune panning illustrated that the number ofGFP-negative, c-kit⁺ cells egressing from the biopsy were 6-fold greatercompared to biopsies cultured alone (FIGS. 15A, 15F). These cells wereCD68^(neg), small, semi-adherent and self-renewing (FIG. 15D, 15H). Asexpected, c-kit⁺ cells could not be harvested from MSC-control cultures.Similarly to our in-situ findings, immunocytochemical analysisdocumented the development of connexin-43 mediated cell-cellinteractions between GFP⁺ and c-kit⁺ cells (FIG. 15G). In contrast,c-kit⁺ cells purified from biopsies cultured without MSCs had a large,antigen-presenting cell-morphology that did not proliferate (FIG. 15E).

In contrast to previous studies, purification of c-kit⁺ CPCs from singlebiopsy samples was dramatically accelerated by co-culture with MSCs, andfacilitated an outgrowth of highly myocardiocytic CPCs; greater than 90%of the cells expressed the cardiac transcription factors Nkx2.5 andGATA-4 and the ATP-binding cassette transporter MDR1 while lackingexpression of the VEGF-receptor, KDR (FIGS. 15A-15K ₁). Interestingly, afraction of the cells stained positive for the LIM-homeodomaintranscription factor Isl-1 (FIG. 15J-15J ₂], a marker that identifies asubset of CPCs during development of the embryonic heart, reported towithdraw from the post-natal heart. Importantly, isl-1 expression couldnot be detected in vivo and was absent from the c-kit+ CPCs found in theadult porcine myocardium. This finding corroborates previous reports,where the post-natal phenotype of c-kit+ CPCs represented a distinctpopulation from the isl-1⁺ CPCs. These observations highlight thecapacity of MSCs to regenerate stem cell niches and signify a key rolefor c-kit and isl-1 that could link mechanisms between cardiacdevelopment and disease.

To test the capacity of the CPCs to differentiate into myocytes,cocultures with neonatal rat cardiomyocytes (NRCMs) were performed.Porcine CPCs were seeded on transwell membranes, and placed on top ofNRCM monolayers. After 3-4 days in co-culture, CPCs differentiated intospontaneously contracting porcine cardiomyocytes, thus exhibiting theircapacity to adopt fully differentiated cardiomyocytic lineages (FIG.15I, FIGS. 17A-17F).

Here, it was demonstrated that extra-cardiac, bone marrow derived MSCs,when injected into hearts following myocardial infarction, facilitatecardiac recovery involving host cells as well as MSC engraftment anddifferentiation. Differentiation of MSCs occurs acutely aftertransplantation, while direct coupling with endogenous c-kit⁺ CPCscauses the latter to amplify and differentiate into fully developedadult cardiomyocytes. This mechanism closely resembles cell-cellinteractions between CPCs and stromal cells in the cardiac niche, whichplay a role for the regulation and expansion of the nascent cardiac stemcell pool. All findings were replicated ex-vivo and developed in-vitrocardiac niches that harbored CPCs previously found only in pre-natalhearts, conveying to important therapeutic potentials.

These findings introduce a novel approach to enhance endogenous cardiacrepair by stimulating the proliferation and maturation of endogenousCPCs. This study illustrates how the hearts' own regenerative propertiescan be stimulated to facilitate cardiac recovery following myocardialinfarction. The approach employed a cell-based rather than pharmacologicapproach to enhance cardiac repair, and as such these results haveimportant biological and therapeutic implications.

Importantly, these experiments have utilized a highly translationalexperimental model of ischemic cardiomyopathy thus reduce to practicemethodologies which could greatly enhance manufacturing of adultcardioblasts from individual hosts. Last, these results offer insightsinto the mechanism of action of a cell-based therapy, wherebysignificant cardiac repair occurs in the absence of a degree ofengraftment and differentiation sufficient to account for the degree ofcardiac functional recovery. Together these findings support the conceptthat MSCs may replenish or restore cardiac stem cell niches lost orinjured during myocardial infarction.

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may be desired and advantageous for any given orparticular application.

The Abstract of the Disclosure is provided to allow the reader toquickly ascertain the nature of the technical disclosure. It issubmitted with the understanding that it will not be used to interpretor limit the scope or meaning of the following claims.

What is claimed is:
 1. A pharmaceutical composition for treating heartdisease comprising mesenchymal stem cells and c-kit⁺ cardiac stem cellsand a pharmaceutically acceptable carrier.
 2. The pharmaceuticalcomposition of claim 1, further comprising cardiac tissue.
 3. Thepharmaceutical composition of claim 1, wherein composition is a solutionwith a concentration of 10-40×10⁶ mesenchymal stem cells/ml.
 4. Thepharmaceutical composition of claim of claim 1, wherein the compositioncomprises 1 million mesenchymal stem cells.
 5. The pharmaceuticalcomposition of claim 1, wherein the composition is in a unit dosage formof 20-50 μl.
 6. The pharmaceutical composition of claim 1, wherein thecomposition is suitable for injection.
 7. The pharmaceutical compositionof claim 1, wherein the carrier comprises phosphate buffered saline(PBS).
 8. The pharmaceutical composition of claim 1, further comprisinga cardiotropic factor.
 9. The pharmaceutical composition of claim 1,wherein the carrier comprises culture media.
 10. The pharmaceuticalcomposition of claim 9, wherein the culture media is serum free.
 11. Thepharmaceutical composition of claim 1, wherein the carrier comprisesphysiological saline.
 12. The pharmaceutical composition of claim 1,wherein the carrier comprises 5% dextrose in water.
 13. Thepharmaceutical composition of claim 1, wherein the heart disease to betreated is cardiomyopathy, hypertrophic cardiomyopathy, dilatedcardiomyopathy, myocarditis, myocardial infarction, or congestive heartfailure.
 14. The pharmaceutical composition of claim 1, wherein theheart disease to be treated is cardiomyopathy.
 15. The pharmaceuticalcomposition of claim 1, wherein the heart disease to be treated ismyocarditis.
 16. The pharmaceutical composition of claim 1, wherein theheart disease to be treated is myocardial infarction.
 17. Thepharmaceutical composition of claim 1, wherein the heart disease to betreated is congestive heart failure.